NPC1L1 (NPC3) and methods of use thereof

ABSTRACT

The present invention provides rat and mouse NPC1L1 polypeptides and polynucleotides encoding the polypeptides. Also provided are methods for detecting agonists and antagonists of NPC1L1. Inhibitors of NPC1L1 can be used for inhibiting intestinal cholesterol absorption in a subject.

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/397,442; filed Jul. 19, 2002 which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention includes NPC1L1 polypeptides andpolynucleotides which encode the polypeptides along with methods of usethereof.

BACKGROUND OF THE INVENTION

[0003] A factor leading to development of vascular disease, a leadingcause of death in industrialized nations, is elevated serum cholesterol.It is estimated that 19% of Americans between the ages of 20 and 74years of age have high serum cholesterol. The most prevalent form ofvascular disease is arteriosclerosis, a condition associated with thethickening and hardening of the arterial wall. Arteriosclerosis of thelarge vessels is referred to as atherosclerosis. Atherosclerosis is thepredominant underlying factor in vascular disorders such as coronaryartery disease, aortic aneurysm, arterial disease of the lowerextremities and cerebrovascular disease.

[0004] Cholesteryl esters are a major component of atheroscleroticlesions and the major storage form of cholesterol in arterial wallcells. Formation of cholesteryl esters is also a step in the intestinalabsorption of dietary cholesterol. Thus, inhibition of cholesteryl esterformation and reduction of serum cholesterol can inhibit the progressionof atherosclerotic lesion formation, decrease the accumulation ofcholesteryl esters in the arterial wall, and block the intestinalabsorption of dietary cholesterol.

[0005] The regulation of whole-body cholesterol homeostasis in mammalsand animals involves the regulation of intestinal cholesterolabsorption, cellular cholesterol trafficking, dietary cholesterol andmodulation of cholesterol biosynthesis, bile acid biosynthesis, steroidbiosynthesis and the catabolism of the cholesterol-containing plasmalipoproteins. Regulation of intestinal cholesterol absorption has provento be an effective means by which to regulate serum cholesterol levels.For example, a cholesterol absorption inhibitor, ezetimibe (

[0006] ), has been shown to be effective in this regard. Identificationof a gene target through which ezetimibe acts is important tounderstanding the process of cholesterol absorption and to thedevelopment of other, novel absorption inhibitors. The present inventionaddresses this need by providing a rat and a mouse homologue of humanNPC1L1 (also known as NPC3; Genbank Accession No. AF192522; Davies, etal., (2000) Genomics 65(2):137-45 and Ioannou, (2000) Mol. Genet.Metab.71(1-2):175-81), the ezetimibe target.

[0007] NPC1L1 is an N-glycosylated protein comprising a YQRL (SEQ ID NO:38) motif (i.e., a trans-golgi network to plasma membrane transportsignal; see Bos, et al., (1993) EMBO J. 12:2219-2228; Humphrey, et al.,(1993) J. Cell. Biol. 120:1123-1135; Ponnambalam, et al., (1994) J.Cell. Biol. 125:253-268 and Rothman, et al., (1996) Science 272:227-234)which exhibits limited tissue distribution and gastrointestinalabundance. Also, the human NPC1L1 promoter includes a Sterol RegulatedElement Binding Protein 1 (SREBP1) binding consensus sequence(Athanikar, et al., (1998) Proc. Natl. Acad. Sci. USA 95:4935-4940;Ericsson, et al., (1996) Proc. Natl. Acad. Sci. USA 93:945-950;Metherall, et al., (1989) J. Biol. Chem. 264:15634-15641; Smith, et al.,(1990) J. Biol. Chem. 265:2306-2310; Bennett, et al., (1999) J. Biol.Chem. 274:13025-13032 and Brown, et al., (1997) Cell 89:331-340). NPC1L1has 42% amino acid sequence homology to human NPC1 (Genbank AccessionNo. AF002020), a receptor responsible for Niemann-Pick C1 disease(Carstea, et al., (1997) Science 277:228-231). Niemann-Pick C1 diseaseis a rare genetic disorder in humans which results in accumulation oflow density lipoprotein (LDL)-derived unesterified cholesterol inlysosomes (Pentchev, et al., (1994) Biochim. Biophys. Acta. 1225:235-243 and Vanier, et al., (1991) Biochim. Biophys. Acta.1096:328-337). In addition, cholesterol accumulates in the trans-golginetwork of npc1⁻ cells, and relocation of cholesterol, to and from theplasma membrane, is delayed. NPC1 and NPC1L1 each possess 13transmembrane spanning segments as well as a sterol-sensing domain(SSD). Several other proteins, including HMG-CoA Reductase (HMG-R),Patched (PTC) and Sterol Regulatory Element Binding ProteinCleavage-Activation Protein (SCAP), include an SSD which is involved insensing cholesterol levels possibly by a mechanism which involves directcholesterol binding (Gil, et al., (1985) Cell 41:249-258; Kumagai, etal., (1995) J. Biol. Chem. 270:19107-19113 and Hua, et al., (1996) Cell87:415-426).

SUMMARY OF THE INVENTION

[0008] The present invention includes an isolated polypeptide comprising42 or more contiguous amino acids from an amino acid sequence selectedfrom SEQ ID NOs: 2 and 12, preferably comprising the amino acid sequenceselected from SEQ ID NOs: 2 and 12. The invention also includes anisolated polynucleotide encoding a polypeptide of SEQ ID NO: 2 or 12,preferably comprising a nucleotide sequence selected from SEQ ID NOs: 1,5-10, 11 and 13. A recombinant vector comprising a polynucleotide of theinvention is also provided along with a host cell comprising the vector.

[0009] The present invention also provides an antibody whichspecifically binds to NPC1L1 (e.g., mouse NPC1L1 or human NPC1L1) or anyantigenic fragment thereof, preferably rat NPC1L1, more preferably apolypeptide comprising an amino acid sequence selected from SEQ ID NO:39-42. Preferably, the antibody is a polyclonal or monoclonal antibody.Preferably, the antibody is obtained from a rabbit.

[0010] The present invention also includes a method for making an NPC1L1polypeptide of the invention comprising culturing a host cell of theinvention under conditions in which the nucleic acid in the cell whichencodes the NPC1L1 polypeptide is expressed. Preferably, the methodincludes the step of isolating the polypeptide from the culture.

[0011] The present invention includes methods for identifying an agonistor antagonist of NPC1L1 comprising (a) contacting a host cell (e.g.,chinese hamster ovary (CHO) cell, a J774 cell, a macrophage cell and aCaco2 cell) expressing a polypeptide comprising the amino acid sequenceof SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 12 or a functionalfragment thereof on a cell surface, in the presence of a known amount ofdetectably labeled (e.g., with ³H or ¹²⁵I) ezetimibe, with a sample tobe tested for the presence of an NPC1L1 agonist or antagonist; and (b)measuring the amount of detectably labeled ezetimibe specifically boundto the polypeptide; wherein an NPC1L1 agonist or antagonist in thesample is identified by measuring substantially reduced binding of thedetectably labeled ezetimibe to the polypeptide, compared to what wouldbe measured in the absence of such an agonist or antagonist.

[0012] Another method for identifying an agonist or antagonist of NPC1L1is also provided. The method comprises (a) placing, in an aqueoussuspension, a plurality of support particles, impregnated with afluorescer (e.g., yttrium silicate, yttrium oxide, diphenyloxazole andpolyvinyltoluene), to which a host cell (e.g., chinese hamster ovary(CHO) cell, a J774 cell, a macrophage cell and a Caco2 cell) expressinga polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQID NO: 4 or SEQ ID NO: 12 or a functional fragment thereof on a cellsurface are attached; (b) adding, to the suspension, radiolabeled (e.g.,with ³H or ¹²⁵I) ezetimibe and a sample to be tested for the presence ofan antagonist or agonist, wherein the radiolabel emits radiation energycapable of activating the fluorescer upon the binding of the ezetimibeto the polypeptide to produce light energy, whereas radiolabeledezetimibe that does not bind to the polypeptide is, generally, too farremoved from the support particles to enable the radioactive energy toactivate the fluorescer; and (c) measuring the light energy emitted bythe fluorescer in the suspension; wherein an NPC1L1 agonist orantagonist in the sample is identified by measuring substantiallyreduced light energy emission, compared to what would be measured in theabsence of such an agonist or antagonist.

[0013] Also provided is a method for identifying an agonist orantagonist of NPC1L1 comprising (a) contacting a host cell (e.g.,chinese hamster ovary (CHO) cell, a J774 cell, a macrophage cell and aCaco2 cell) expressing an polypeptide comprising an amino acid sequenceof SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 12 or a functionalfragment thereof on a cell surface with detectably labeled (e.g., with³H and ¹²⁵I) cholesterol and with a sample to be tested for the presenceof an antagonist or agonist; and (b) measuring the amount of detectablylabeled cholesterol in the cell; wherein an NPC1L1 antagonist in thesample is identified by measuring substantially reduced detectablylabeled cholesterol within the host cell, compared to what would bemeasured in the absence of such an antagonist and wherein an NPC1L1agonist in the sample is identified by measuring substantially increaseddetectably labeled cholesterol within the host cell, compared to whatwould be measured in the absence of such an agonist.

[0014] Also included in the present invention is a mutant mousecomprising a homozygous or heterozygous disruption of endogenous,chromosomal NPC1L1 wherein, preferably, the mouse does not produce anyfunctional NPC1L1 protein.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention includes an NPC1L1 polypeptide from rat andfrom mouse along with polynucleotides encoding the respectivepolypeptides. Preferably, the rat NPC1L1 polypeptide comprises the aminoacid sequence set forth in SEQ ID NO: 2 and the mouse NPC1L1 polypeptidecomprises the amino acid sequence set forth in SEQ ID NO.12. The ratNPC1L1 polynucleotide of SEQ ID NO:1 or 10 encodes the rat NPC1L1polypeptide. The mouse NPC1L1 polynucleotide of SEQ ID NO:11 or 13encodes the mouse NPC1L1 polypeptide.

[0016] The present invention includes any polynucleotide or polypeptidecomprising a nucleotide or amino acid sequence referred to, below, inTable 1. TABLE 1 Polynucleotides and Polypeptides of the Invention.Polynucleotide or Polypeptide Sequence Identifier Rat NPC1L1polynucleotide SEQ ID NO: 1 Rat NPC1L1 polypeptide SEQ ID NO: 2 HumanNPC1L1 polynucleotide SEQ ID NO: 3 Human NPC1L1 polypeptide SEQ ID NO: 4Rat NPC1L1 expressed sequence tag SEQ ID NO: 5 603662080F1 (partialsequence) Rat NPC1L1 expressed sequence tag SEQ ID NO: 6 603665037F1(partial sequence) Rat NPC1L1 expressed sequence tag SEQ ID NO: 7604034587F1 (partial sequence) EST 603662080F1 with downstream SEQ IDNO: 8 sequences added EST 603662080F1 with upstream and SEQ ID NO: 9downstream sequences added Back-translated polynucleotide sequence ofSEQ ID NO: 10 rat NPC1L1 Mouse NPC1L1 polynucleotide SEQ ID NO: 11 MouseNPC1L1 polypeptide SEQ ID NO: 12 Back-translated polynucleotide sequenceof SEQ ID NO: 13 mouse NPC1L1

[0017] A human NPC1L1 is also disclosed under Genbank Accession NumberAF192522. As discussed below, the nucleotide sequence of the rat NPC1L1set forth in SEQ ID NO: 1 was obtained from an expressed sequence tag(EST) from a rat jejunum enterocyte cDNA library. SEQ ID NOs: 5-7include partial nucleotide sequences of three independent cDNA clones.The downstream sequence of the SEQ ID NO: 5 EST (603662080F1) weredetermined; the sequencing data from these experiments are set forth inSEQ ID NO: 8. The upstream sequences were also determined; these dataare set forth in SEQ ID NO: 9.

[0018] SEQ ID NOs: 43 and 44 are the nucleotide and amino acid sequence,respectively, of human NPC1L1 which is disclosed under Genbank AccessionNo.: AF192522 (see Davies, et al., (2000) Genomics 65(2):137-45).

[0019] SEQ ID NO: 45 is the nucleotide sequence of a mouse NPC1L1 whichis disclosed under Genbank Accession No. AK078947.

Molecular Biology

[0020] In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (11989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook, et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1985)); Transcription And Translation (B. D. Hames & S. J. Higgins,eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel, et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(11994).

[0021] The back-translated sequences of SEQ ID NO: 10 and of SEQ ID NO:13 uses the single-letter code shown in Table 1 of Annex C, Appendix 2of the PCT Administrative Instruction in the Manual of PatentExamination Procedure.

[0022] A “polynucleotide”, “nucleic acid” or “nucleic acid molecule” mayrefer to the phosphate ester polymeric form of ribonucleosides(adenosine, guanosine, uridine or cytidine; “RNA molecules”) ordeoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, ordeoxycytidine; “DNA molecules”), or any phosphoester analogs thereof,such as phosphorothioates and thioesters, in single stranded form,double-stranded form or otherwise.

[0023] A “polynucleotide sequence”, “nucleic acid sequence” or“nucleotide sequence” is a series of nucleotide bases (also called“nucleotides”) in a nucleic acid, such as DNA or RNA, and means anychain of two or more nucleotides.

[0024] A “coding sequence” or a sequence “encoding” an expressionproduct, such as a RNA, polypeptide, protein, or enzyme, is a nucleotidesequence that, when expressed, results in production of the product.

[0025] The term “gene” means a DNA sequence that codes for orcorresponds to a particular sequence of ribonucleotides or amino acidswhich comprise all or part of one or more RNA molecules, proteins orenzymes, and may or may not include regulatory DNA sequences, such aspromoter sequences, which determine, for example, the conditions underwhich the gene is expressed. Genes may be transcribed from DNA to RNAwhich may or may not be translated into an amino acid sequence.

[0026] The present invention includes nucleic acid fragments of any ofSEQ ID NOs: 1, 5-11 or 13. A nucleic acid “fragment” includes at leastabout 30 (e.g., 31, 32, 33, 34), preferably at least about 35 (e.g, 25,26, 27, 28, 29, 30, 31, 32, 33 or 34), more preferably at least about 45(e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44), and most preferably atleast about 126 or more contiguous nucleotides (e.g., 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 150, 160, 170, 180, 190, 200,300, 400, 500, 1000 or 1200) from any of SEQ ID NOs: 1, 5-11 or 13.

[0027] The present invention also includes nucleic acid fragmentsconsisting of at least about 7 (e.g., 9, 12, 17, 19), preferably atleast about 20 (e.g., 30, 40, 50, 60), more preferably about 70 (e.g.,80, 90, 95), yet more preferably at least about 100 (e.g., 105, 110,114) and even more preferably at least about 115 (e.g., 117, 119, 120,122, 124, 125, 126) contiguous nucleotides from any of SEQ ID NOs: 1,5-11 or 13.

[0028] As used herein, the term “oligonucleotide” refers to a nucleicacid, generally of no more than about 100 nucleotides (e.g., 30, 40, 50,60, 70, 80, or 90), that may be hybridizable to a genomic DNA molecule,a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, orother nucleic acid of interest. Oligonucleotides can be labeled, e.g.,by incorporation of ³²P-nucleotides, ³H-nucleotides, ¹⁴C-nucleotides,³⁵S-nucleotides or nucleotides to which a label, such as biotin, hasbeen covalently conjugated. In one embodiment, a labeled oligonucleotidecan be used as a probe to detect the presence of a nucleic acid. Inanother embodiment, oligonucleotides (one or both of which may belabeled) can be used as PCR primers, either for cloning full length or afragment of the gene, or to detect the presence of nucleic acids.Generally, oligonucleotides are prepared synthetically, preferably on anucleic acid synthesizer.

[0029] A “protein sequence”, “peptide sequence” or “polypeptidesequence” or “amino acid sequence” may refer to a series of two or moreamino acids in a protein, peptide or polypeptide.

[0030] “Protein”, “peptide” or “polypeptide” includes a contiguousstring of two or more amino acids Preferred peptides of the inventioninclude those set forth in any of SEQ ID NOs: 2 or 12 as well asvariants and fragments thereof. Such fragments preferably comprise atleast about 10 (e.g., 11, 12, 13, 14, 15, 16, 17, 18 or 19), morepreferably at least about 20 (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40), and yet more preferably at least about 42 (e.g., 43, 44,45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120 or 130) or morecontiguous amino acid residues from any of SEQ ID NOs: 2 or 12.

[0031] The present invention also includes polypeptides, preferablyantigenic polypeptides, consisting of at least about 7 (e.g., 9, 10, 13,15, 17, 19), preferably at least about 20 (e.g., 22, 24, 26, 28), yetmore preferably at least about 30 (e.g., 32, 34, 36, 38) and even morepreferably at least about 40 (e.g., 41, 42) contiguous amino acids fromany of SEQ ID NOs: 2 or 12.

[0032] The polypeptides of the invention can be produced by proteolyticcleavage of an intact peptide, by chemical synthesis or by theapplication of recombinant DNA technology and are not limited topolypeptides delineated by proteolytic cleavage sites. The polypeptides,either alone or cross-linked or conjugated to a carrier molecule torender them more immunogenic, are useful as antigens to elicit theproduction of antibodies and fragments thereof. The antibodies can beused, e.g., in immunoassays for immunoaffinity purification or forinhibition of NPC1L1, etc.

[0033] The terms “isolated polynucleotide” or “isolated polypeptide”include a polynucleotide (e.g., RNA or DNA molecule, or a mixed polymer)or a polypeptide, respectively, which are partially or fully separatedfrom other components that are normally found in cells or in recombinantDNA expression systems. These components include, but are not limitedto, cell membranes, cell walls, ribosomes, polymerases, serum componentsand extraneous genomic sequences.

[0034] An isolated polynucleotide or polypeptide will, preferably, be anessentially homogeneous composition of molecules but may contain someheterogeneity.

[0035] “Amplification” of DNA as used herein may denote the use ofpolymerase chain reaction (PCR) to increase the concentration of aparticular DNA sequence within a mixture of DNA sequences. For adescription of PCR see Saiki, et al., Science (1988) 239:487.

[0036] The term “host cell” includes any cell of any organism that isselected, modified, transfected, transformed, grown, or used ormanipulated in any way, for the production of a substance by the cell,for example the expression or replication, by the cell, of a gene, a DNAor RNA sequence or a protein. Preferred host cells include chinesehamster ovary (CHO) cells, murine macrophage J774 cells or any othermacrophage cell line and human intestinal epithelial Caco2 cells.

[0037] The nucleotide sequence of a nucleic acid may be determined byany method known in the art (e.g., chemical sequencing or enzymaticsequencing). “Chemical sequencing” of DNA includes methods such as thatof Maxam and Gilbert (1977) (Proc. Natl. Acad. Sci. USA 74:560), inwhich DNA is randomly cleaved using individual base-specific reactions.“Enzymatic sequencing” of DNA includes methods such as that of Sanger(Sanger, et al., (1977) Proc. Natl. Acad. Sci. USA 74:5463).

[0038] The nucleic acids herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like.

[0039] In general, a “promoter” or “promoter sequence” is a DNAregulatory region capable of binding an RNA polymerase in a cell (e.g.,directly or through other promoter-bound proteins or substances) andinitiating transcription of a coding sequence. A promoter sequence is,in general, bounded at its 3′ terminus by the transcription initiationsite and extends upstream (5′ direction) to include the minimum numberof bases or elements necessary to initiate transcription at any level.Within the promoter sequence may be found a transcription initiationsite (conveniently defined, for example, by mapping with nuclease S1),as well as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. The promoter may be operably associatedwith other expression control sequences, including enhancer andrepressor sequences or with a nucleic acid of the invention. Promoterswhich may be used to control gene expression include, but are notlimited to, cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and5,168,062), the SV40 early promoter region (Benoist, et al., (1981)Nature 290:304-310), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., (1980) Cell 22:787-797),the herpes thymidine kinase promoter (Wagner, et al., (1981) Proc. Natl.Acad. Sci. USA 78:1441-1445), the regulatory sequences of themetallothionein gene (Brinster, et al., (1982) Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Komaroff, et al., (1978) Proc. Natl. Acad. Sci. USA75:3727-3731), or the tac promoter (DeBoer, et al., (1983) Proc. Natl.Acad. Sci. USA 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American (1980) 242:74-94; and promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or thealkaline phosphatase promoter.

[0040] A coding sequence is “under the control of”, “functionallyassociated with” or “operably associated with” transcriptional andtranslational control sequences in a cell when the sequences direct RNApolymerase mediated transcription of the coding sequence into RNA,preferably mRNA, which then may be RNA spliced (if it contains introns)and, optionally, translated into a protein encoded by the codingsequence.

[0041] The terms “express” and “expression” mean allowing or causing theinformation in a gene, RNA or DNA sequence to become manifest; forexample, producing a protein by activating the cellular functionsinvolved in transcription and translation of a corresponding gene. A DNAsequence is expressed in or by a cell to form an “expression product”such as an RNA (e.g., mRNA) or a protein. The expression product itselfmay also be said to be “expressed” by the cell.

[0042] The term “transformation” means the introduction of a nucleicacid into a cell. The introduced gene or sequence may be called a“clone”. A host cell that receives the introduced DNA or RNA has been“transformed” and is a “transformant” or a “clone.” The DNA or RNAintroduced to a host cell can come from any source, including cells ofthe same genus or species as the host cell, or from cells of a differentgenus or species.

[0043] The term “vector” includes a vehicle (e.g., a plasmid) by which aDNA or RNA sequence can be introduced into a host cell, so as totransform the host and, optionally, promote expression and/orreplication of the introduced sequence.

[0044] Vectors that can be used in this invention include plasmids,viruses, bacteriophage, integratable DNA fragments, and other vehiclesthat may facilitate introduction of the nucleic acids into the genome ofthe host. Plasmids are the most commonly used form of vector but allother forms of vectors which serve a similar function and which are, orbecome, known in the art are suitable for use herein. See, e.g.,Pouwels, et al., Cloning Vectors: A Laboratory Manual, 1985 andSupplements, Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses 1988, Buttersworth,Boston, Mass.

[0045] The term “expression system” means a host cell and compatiblevector which, under suitable conditions, can express a protein ornucleic acid which is carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, insect host cells and Baculovirus vectors, and mammalian hostcells and vectors.

[0046] Expression of nucleic acids encoding the NPC1L1 polypeptides ofthis invention can be carried out by conventional methods in eitherprokaryotic or eukaryotic cells. Although E. coli host cells areemployed most frequently in prokaryotic systems, many other bacteria,such as various strains of Pseudomonas and Bacillus, are known in theart and can be used as well. Suitable host cells for expressing nucleicacids encoding the NPC1L1 polypeptides include prokaryotes and highereukaryotes. Prokaryotes include both gram-negative and gram-positiveorganisms, e.g., E. coli and B. subtilis. Higher eukaryotes includeestablished tissue culture cell lines from animal cells, both ofnon-mammalian origin, e.g., insect cells, and birds, and of mammalianorigin, e.g., human, primates, and rodents.

[0047] Prokaryotic host-vector systems include a wide variety of vectorsfor many different species. A representative vector for amplifying DNAis pBR322 or many of its derivatives (e.g., pUC18 or 19). Vectors thatcan be used to express the NPC1L1 polypeptides include, but are notlimited to, those containing the lac promoter (pUC-series); trp promoter(pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pR promoters(pOTS); or hybrid promoters such as ptac (pDR540). See Brosius et al.,“Expression Vectors Employing Lambda-, trp-, lac-, and Ipp-derivedPromoters”, in Rodriguez and Denhardt (eds.) Vectors: A Survey ofMolecular Cloning Vectors and Their Uses, 1988, Buttersworth, Boston,pp. 205-236. Many polypeptides can be expressed, at high levels, in anE. coli/T7 expression system as disclosed in U.S. Pat. Nos. 4,952,496,5,693,489 and 5,869,320 and in Davanloo, P., et al., (1984) Proc. Natl.Acad. Sci. USA 81: 2035-2039; Studier, F. W., et al., (1986) J. Mol.Biol. 189: 113-130; Rosenberg, A. H., et al., (1987) Gene 56: 125-135;and Dunn, J. J., et al., (1988) Gene 68: 259.

[0048] Higher eukaryotic tissue culture cells may also be used for therecombinant production of the NPC1L1 polypeptides of the invention.Although any higher eukaryotic tissue culture cell line might be used,including insect baculovirus expression systems, mammalian cells arepreferred. Transformation or transfection and propagation of such cellshave become a routine procedure. Examples of useful cell lines includeHeLa cells, chinese hamster ovary (CHO) cell lines, J774 cells, Caco2cells, baby rat kidney (BRK) cell lines, insect cell lines, bird celllines, and monkey (COS) cell lines. Expression vectors for such celllines usually include an origin of replication, a promoter, atranslation initiation site, RNA splice sites (if genomic DNA is used),a polyadenylation site, and a transcription termination site. Thesevectors also, usually, contain a selection gene or amplification gene.Suitable expression vectors may be plasmids, viruses, or retrovirusescarrying promoters derived, e.g., from such sources as adenovirus, SV40,parvoviruses, vaccinia virus, or cytomegalovirus. Examples of expressionvectors include pCR®3.1, pcDNA1, pCD (Okayama, et al., (1985) Mol. CellBiol. 5:1136), pMC1neo Poly-A (Thomas, et al., (1987) Cell 51:503),pREP8, pSVSPORT and derivatives thereof, and baculovirus vectors such aspAC373 or pAC610. One embodiment of the invention includes membranebound NPC1L1. In this embodiment, NPC1L1 can be expressed in the cellmembrane of a eukaryotic cell and the membrane bound protein can beisolated from the cell by conventional methods which are known in theart.

[0049] The present invention also includes fusions which include theNPC1L1 polypeptides and NPC1L1 polynucleotides of the present inventionand a second polypeptide or polynucleotide moiety, which may be referredto as a “tag”. The fusions of the present invention may comprise any ofthe polynucleotides or polypeptides set forth in Table 1 or anysubsequence or fragment thereof (discussed above). The fusedpolypeptides of the invention may be conveniently constructed, forexample, by insertion of a polynucleotide of the invention or fragmentthereof into an expression vector. The fusions of the invention mayinclude tags which facilitate purification or detection. Such tagsinclude glutathione-S-transferase (GST), hexahistidine (His6) tags,maltose binding protein (MBP) tags, haemagglutinin (HA) tags, cellulosebinding protein (CBP) tags and myc tags. Detectable tags such as ³²P,³⁵S, ³H, ^(99m)Tc, ¹²³I, ¹¹¹In, ⁶⁸Ga, ¹⁸F, ¹²⁵I, ¹³¹I, ^(113m)In, ⁷⁶Br,⁶⁷Ga, ^(99m)Tc, ¹²³I, ¹¹¹In and ⁶⁸Ga may also be used to label thepolypeptides and polynucleotides of the invention. Methods forconstructing and using such fusions are very conventional and well knownin the art.

[0050] Modifications (e.g., post-translational modifications) that occurin a polypeptide often will be a function of how it is made. Forpolypeptides made by expressing a cloned gene in a host, for instance,the nature and extent of the modifications, in large part, will bedetermined by the host cell's post-translational modification capacityand the modification signals present in the polypeptide amino acidsequence. For instance, as is well known, glycosylation often does notoccur in bacterial hosts such as E. coli. Accordingly, whenglycosylation is desired, a polypeptide can be expressed in aglycosylating host, generally a eukaryotic cell. Insect cells oftencarry out post-translational glycosylations which are similar to thoseof mammalian cells. For this reason, insect cell expression systems havebeen developed to express, efficiently, mammalian proteins having nativepatterns of glycosylation. An insect cell which may be used in thisinvention is any cell derived from an organism of the class Insecta.Preferably, the insect is Spodoptera fruigiperda (Sf9 or Sf21) orTrichoplusia ni (High 5). Examples of insect expression systems that canbe used with the present invention, for example to produce NPC1L1polypeptide, include Bac-To-Bac (Invitrogen Corporation, Carlsbad,Calif.) or Gateway (Invitrogen Corporation, Carlsbad, Calif.). Ifdesired, deglycosylation enzymes can be used to remove carbohydratesattached during production in eukaryotic expression systems.

[0051] Other modifications may also include addition of aliphatic estersor amides to the polypeptide carboxyl terminus. The present inventionalso includes analogs of the NPC1L1 polypeptides which containmodifications, such as incorporation of unnatural amino acid residues,or phosphorylated amino acid residues such as phosphotyrosine,phosphoserine or phosphothreonine residues. Other potentialmodifications include sulfonation, biotinylation, or the addition ofother moieties. For example, the NPC1L1 polypeptides of the inventionmay be appended with a polymer which increases the half-life of thepeptide in the body of a subject. Preferred polymers includepolyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa and 40 kDa), dextran andmonomethoxypolyethylene glycol (mPEG).

[0052] The peptides of the invention may also be cyclized. Specifically,the amino- and carboxy-terminal residues of an NPC1L1 polypeptide or twointernal residues of an NPC1L1 polypeptide of the invention can be fusedto create a cyclized peptide. Methods for cyclizing peptides areconventional and very well known in the art; for example see Gurrath, etal., (1992) Eur. J. Biochem. 210:911-921.

[0053] The present invention contemplates any superficial or slightmodification to the amino acid or nucleotide sequences which correspondto the polypeptides of the invention. In particular, the presentinvention contemplates sequence conservative variants of the nucleicacids which encode the polypeptides of the invention.“Sequence-conservative variants” of a polynucleotide sequence are thosein which a change of one or more nucleotides in a given codon results inno alteration in the amino acid encoded at that position.Function-conservative variants of the polypeptides of the invention arealso contemplated by the present invention. “Function-conservativevariants” are those in which one or more amino acid residues in aprotein or enzyme have been changed without altering the overallconformation and function of the polypeptide, including, but, by nomeans, limited to, replacement of an amino acid with one having similarproperties. Amino acids with similar properties are well known in theart. For example, polar/hydrophilic amino acids which may beinterchangeable include asparagine, glutamine, serine, cysteine,threonine, lysine, arginine, histidine, aspartic acid and glutamic acid;nonpolar/hydrophobic amino acids which may be interchangeable includeglycine, alanine, valine, leucine, isoleucine, proline, tyrosine,phenylalanine, tryptophan and methionine; acidic amino acids which maybe interchangeable include aspartic acid and glutamic acid and basicamino acids which may be interchangeable include histidine, lysine andarginine.

[0054] The present invention includes polynucleotides encoding rat ormouse NPC1L1 and fragments thereof as well as nucleic acids whichhybridize to the polynucleotides. Preferably, the nucleic acidshybridize under low stringency conditions, more preferably undermoderate stringency conditions and most preferably under high stringencyconditions. A nucleic acid molecule is “hybridizable” to another nucleicacid molecule, such as a cDNA, genomic DNA, or RNA, when a singlestranded form of the nucleic acid molecule can anneal to the othernucleic acid molecule under the appropriate conditions of temperatureand solution ionic strength (see Sambrook, et al., supra). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. Typical low stringency hybridization conditionsare 55° C., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide at 42° C.; or30% formamide, 5×SSC, 0.5% SDS at 42° C. Typical, moderate stringencyhybridization conditions are similar to the low stringency conditionsexcept the hybridization is carried out in 40% formamide, with 5× or6×SSC at 42° C. High stringency hybridization conditions are similar tolow stringency conditions except the hybridization conditions arecarried out in 50% formamide, 5× or 6×SSC and, optionally, at a highertemperature (e.g., higher than 42° C.: 57° C., 59° C., 60° C., 62° C.,63° C., 65° C. or 68° C.). In general, SSC is 0.15M NaCl and 0.015MNa-citrate. Hybridization requires that the two nucleic acids containcomplementary sequences, although, depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation, variables well known inthe art. The greater the degree of similarity or homology between twonucleotide sequences, the higher the stringency under which the nucleicacids may hybridize. For hybrids of greater than 100 nucleotides inlength, equations for calculating the melting temperature have beenderived (see Sambrook, et al., supra, 9.50-9.51). For hybridization withshorter nucleic acids, i.e., oligonucleotides, the position ofmismatches becomes more important, and the length of the oligonucleotidedetermines its specificity (see Sambrook, et al., supra).

[0055] Also included in the present invention are polynucleotidescomprising nucleotide sequences and polypeptides comprising amino acidsequences which are at least about 70% identical, preferably at leastabout 80% identical, more preferably at least about 90% identical andmost preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%,99%, 100%) to the reference rat NPC1L1 nucleotide (e.g., any of SEQ IDNOs: 1 or 5-10) and amino acid sequences (e.g., SEQ ID NO: 2) or themouse NPC1L1 nucleotide (e.g., any of SEQ ID NOs: 11 or 13) and aminoacids sequences (e.g., SEQ ID NO: 12), when the comparison is performedby a BLAST algorithm wherein the parameters of the algorithm areselected to give the largest match between the respective sequences overthe entire length of the respective reference sequences. Polypeptidescomprising amino acid sequences which are at least about 70% similar,preferably at least about 80% similar, more preferably at least about90% similar and most preferably at least about 95% similar (e.g., 95%,96%, 97%, 98%, 99%, 100%) to the reference rat NPC1L1 amino acidsequence of SEQ ID NO: 2 or the mouse NPC1L1 amino acid sequence of SEQID NO: 12, when the comparison is performed with a BLAST algorithmwherein the parameters of the algorithm are selected to give the largestmatch between the respective sequences over the entire length of therespective reference sequences, are also included in the presentinvention.

[0056] Sequence identity refers to exact matches between the nucleotidesor amino acids of two sequences which are being compared. Sequencesimilarity refers to both exact matches between the amino acids of twopolypeptides which are being compared in addition to matches betweennonidentical, biochemically related amino acids. Biochemically relatedamino acids which share similar properties and may be interchangeableare discussed above.

[0057] The following references regarding the BLAST algorithm are hereinincorporated by reference: BLAST ALGORITHMS: Altschul, S. F., et al.,(1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet.3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol. 266:131-141;Altschul, S. F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang,J., et al., (1997) Genome Res. 7:649-656; Wootton, J. C., et al., (1993)Comput. Chem. 17:149-163; Hancock, J. M., et al., (1994) Comput. Appl.Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “Amodel of evolutionary change in proteins.” in Atlas of Protein Sequenceand Structure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp.345-352, Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M.,et al., “Matrices for detecting distant relationships.” in Atlas ofProtein Sequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff(ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.;Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J., etal., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl.Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al., (1993) J. Mol.Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc.Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, New York.

Protein Purification

[0058] The proteins, polypeptides and antigenic fragments of thisinvention can be purified by standard methods, including, but notlimited to, salt or alcohol precipitation, affinity chromatography(e.g., used in conjunction with a purification tagged NPC1L1 polypeptideas discussed above), preparative disc-gel electrophoresis, isoelectricfocusing, high pressure liquid chromatography (HPLC), reversed-phaseHPLC, gel filtration, cation and anion exchange and partitionchromatography, and countercurrent distribution. Such purificationmethods are well known in the art and are disclosed, e.g., in “Guide toProtein Purification”, Methods in Enzymology, Vol. 182, M. Deutscher,Ed., 1990, Academic Press, New York, N.Y.

[0059] Purification steps can be followed by performance of assays forreceptor binding activity as described below. Particularly where anNPC1L1 polypeptide is being isolated from a cellular or tissue source,it is preferable to include one or more inhibitors of proteolyticenzymes in the assay system, such as phenylmethanesulfonyl fluoride(PMSF), Pefabloc SC, pepstatin, leupeptin, chymostatin and EDTA.

Antibody Molecules

[0060] Antigenic (including immunogenic) fragments of the NPC1L1polypeptides of the invention are within the scope of the presentinvention (e.g., 42 or more contiguous amino acids from SEQ ID NO: 2, 4or 12). The antigenic peptides may be useful, inter alia, for preparingantibody molecules which recognize NPC1L1. Anti-NPC1L1 antibodymolecules are useful NPC1L1 antagonists.

[0061] An antigen is any molecule that can bind specifically to anantibody. Some antigens cannot, by themselves, elicit antibodyproduction. Those that can induce antibody production are immunogens.

[0062] Preferably, anti-NPC1L1 antibodies recognize an antigenic peptidecomprising an amino acid sequence selected from SEQ ID NOs: 39-42 (e.g.,an antigen derived from rat NPC1L1). More preferably, the antibody isA0715, A0716, A0717, A0718, A0867, A0868, A1801 or A1802.

[0063] The term “antibody molecule” includes, but is not limited to,antibodies and fragments (preferably antigen-binding fragments) thereof.The term includes monoclonal antibodies, polyclonal antibodies,bispecific antibodies, Fab antibody fragments, F(ab)₂ antibodyfragments, Fv antibody fragments (e.g., V_(H) or V_(L)), single chain Fvantibody fragments and dsFv antibody fragments. Furthermore, theantibody molecules of the invention may be fully human antibodies, mouseantibodies, rat antibodies, rabbit antibodies, goat antibodies, chickenantibodies, humanized antibodies or chimeric antibodies.

[0064] Although it is not always necessary, when NPC1L1 polypeptides areused as antigens to elicit antibody production in an immunologicallycompetent host, smaller antigenic fragments are, preferably, firstrendered more immunogenic by cross-linking or concatenation, or bycoupling to an immunogenic carrier molecule (i.e., a macromoleculehaving the property of independently eliciting an immunological responsein a host animal, such as diptheria toxin or tetanus). Cross-linking orconjugation to a carrier molecule may be required because smallpolypeptide fragments sometimes act as haptens (molecules which arecapable of specifically binding to an antibody but incapable ofeliciting antibody production, i.e., they are not immunogenic).Conjugation of such fragments to an immunogenic carrier molecule rendersthem more immunogenic through what is commonly known as the “carriereffect”.

[0065] Carrier molecules include, e.g., proteins and natural orsynthetic polymeric compounds such as polypeptides, polysaccharides,lipopolysaccharides etc. Protein carrier molecules are especiallypreferred, including, but not limited to, keyhole limpet hemocyanin andmammalian serum proteins such as human or bovine gammaglobulin, human,bovine or rabbit serum albumin, or methylated or other derivatives ofsuch proteins. Other protein carriers will be apparent to those skilledin the art. Preferably, the protein carrier will be foreign to the hostanimal in which antibodies against the fragments are to be elicited.

[0066] Covalent coupling to the carrier molecule can be achieved usingmethods well known in the art, the exact choice of which will bedictated by the nature of the carrier molecule used. When theimmunogenic carrier molecule is a protein, the fragments of theinvention can be coupled, e.g., using water-soluble carbodiimides suchas dicyclohexylcarbodiimide or glutaraldehyde.

[0067] Coupling agents, such as these, can also be used to cross-linkthe fragments to themselves without the use of a separate carriermolecule. Such cross-linking into aggregates can also increaseimmunogenicity. Immunogenicity can also be increased by the use of knownadjuvants, alone or in combination with coupling or aggregation.

[0068] Adjuvants for the vaccination of animals include, but are notlimited to, Adjuvant 65 (containing peanut oil, mannide monooleate andaluminum monostearate); Freund's complete or incomplete adjuvant;mineral gels such as aluminum hydroxide, aluminum phosphate and alum;surfactants such as hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′,N′-bis(2-hydroxymethyl) propanediamine,methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran,dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides suchas muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.The polypeptides could also be administered following incorporation intoliposomes or other microcarriers.

[0069] Information concerning adjuvants and various aspects ofimmunoassays are disclosed, e.g., in the series by P. Tijssen, Practiceand Theory of Enzyme Immunoassays, 3rd Edition, 1987, Elsevier, NewYork. Other useful references covering methods for preparing polyclonalantisera include Microbiology, 1969, Hoeber Medical Division, Harper andRow; Landsteiner, Specificity of Serological Reactions, 1962, DoverPublications, New York, and Williams, et al., Methods in Immunology andImmunochemistry, Vol. 1, 1967, Academic Press, New York.

[0070] The anti-NPC1L1 antibody molecules of the invention preferablyrecognize human, mouse or rat NPC1L1; however, the present inventionincludes antibody molecules which recognize NPC1L1 from any species,preferably mammals (e.g., cat, sheep or horse). The present inventionalso includes complexes comprising an NPC1L1 polypeptide of theinvention and an anti-NPC1L1 antibody molecule. Such complexes can bemade by simply contacting the antibody molecule with its cognatepolypeptide.

[0071] Various methods may be used to make the antibody molecules of theinvention. Human antibodies can be made, for example, by methods whichare similar to those disclosed in U.S. Pat. Nos. 5,625,126; 5,877,397;6,255,458; 6,023,010 and 5,874,299.

[0072] Hybridoma cells which produce the monoclonal anti-NPC1L1antibodies may be produced by methods which are commonly known in theart. These methods include, but are not limited to, the hybridomatechnique originally developed by Kohler, et al., (I 975) (Nature256:495-497), as well as the trioma technique (Hering, et al., (1988)Biomed. Biochim. Acta. 47:211-216 and Hagiwara, et al., (1993) Hum.Antibod. Hybridomas 4:15), the human B-cell hybridoma technique (Kozbor,et al., (1983) Immunology Today 4:72 and Cote, et al., (1983) Proc.Natl. Acad. Sci. U.S.A 80:2026-2030), and the EBV-hybridoma technique(Cole, et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96, 1985). ELISA may be used to determine ifhybridoma cells are expressing anti-NPC1L1 antibodies.

[0073] The anti-NPC1L1 antibody molecules of the present invention mayalso be produced recombinantly (e.g., in an E. coli T7 expression systemas discussed above). In this embodiment, nucleic acids encoding theantibody molecules of the invention (e.g., V_(H) or V_(L)) may beinserted into a pet-based plasmid and expressed in the E. coli/T7system. There are several methods by which to produce recombinantantibodies which are known in the art. An example of a method forrecombinant production of antibodies is disclosed in U.S. Pat. No.4,816,567. See also Skerra, A., et al., (1988) Science 240:1038-1041;Better, M., et al., (1988) Science 240:1041-1043 and Bird, R. E., etal., (1988) Science 242:423-426.

[0074] The term “monoclonal antibody,” includes an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible, naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Monoclonal antibodies are advantageousin that they may be synthesized by a hybridoma culture, essentiallyuncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. The monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method as described byKohler, et al., (1975) Nature 256:495.

[0075] The term “polyclonal antibody” includes an antibody which wasproduced among or in the presence of one or more other, non-identicalantibodies. In general, polyclonal antibodies are produced from aB-lymphocyte in the presence of several other B-lymphocytes whichproduced non-identical antibodies. Typically, polyclonal antibodies areobtained directly from an immunized animal (e.g., a rabbit).

[0076] A “bispecific antibody” comprises two different antigen bindingregions which bind to distinct antigens. Bispecific antibodies, as wellas methods of making and using the antibodies, are conventional and verywell known in the art.

[0077] Anti-idiotypic antibodies or anti-idiotypes are antibodiesdirected against the antigen-combining region or variable region (calledthe idiotype) of another antibody molecule. As disclosed by Jerne(Jerne, N. K., (1974) Ann. Immunol. (Paris) 125c:373 and Jerne, N. K.,et al., (1982) EMBO 1:234), immunization with an antibody moleculeexpressing a paratope (antigen-combining site) for a given antigen(e.g., NPC1L1) will produce a group of anti-antibodies, some of whichshare, with the antigen, a complementary structure to the paratope.Immunization with a subpopulation of the anti-idiotypic antibodies will,in turn, produce a subpopulation of antibodies or immune cell subsetsthat are reactive to the initial antigen.

[0078] The term “fully human antibody” refers to an antibody whichcomprises human immunoglobulin sequences only. Similarly, “mouseantibody” refers to an antibody which comprises mouse immunoglobulinsequences only.

[0079] “Human/mouse chimeric antibody” refers to an antibody whichcomprises a mouse variable region (V_(H) and V_(L)) fused to a humanconstant region.

[0080] “Humanized” anti-NPC1L1 antibodies are also within the scope ofthe present invention. Humanized forms of non-human (e.g., murine)antibodies are chimeric immunoglobulins, which contain minimal sequencederived from non-human immunoglobulin. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region of the recipient arereplaced by residues from a complementary determining region of anonhuman species (donor antibody), such as mouse, rat or rabbit, havinga desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are also replaced bycorresponding non-human residues.

[0081] “Single-chain Fv” or “sFv” antibody fragments include the V_(H)and/or V_(L) domains of an antibody, wherein these domains are presentin a single polypeptide chain. Generally, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding.Techniques described for the production of single chain antibodies (U.S.Pat. Nos. 5,476,786; 5,132,405 and 4,946,778) can be adapted to produceanti-NPC1L1 specific, single chain antibodies. For a review of sFv seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds. Springer-Verlag, N.Y., pp. 269-315 (1994).

[0082] “Disulfide stabilized Fv fragments” and “dsFv” include moleculeshaving a variable heavy chain (V_(H)) and/or a variable light chain(V_(L)) which are linked by a disulfide bridge.

[0083] Antibody fragments within the scope of the present invention alsoinclude F(ab)₂ fragments which may be produced by enzymatic cleavage ofan IgG by, for example, pepsin. Fab fragments may be produced by, forexample, reduction of F(ab)₂ with dithiothreitol or mercaptoethylamine.

[0084] An Fv fragment is a V_(L) or V_(H) region.

[0085] Depending on the amino acid sequences of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are at least five major classes of immunoglobulins: IgA,IgD, IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 andIgA-2.

[0086] The anti-NPC1L1 antibody molecules of the invention may also beconjugated to a chemical moiety. The chemical moiety may be, inter alia,a polymer, a radionuclide or a cytotoxic factor. Preferably, thechemical moiety is a polymer which increases the half-life of theantibody molecule in the body of a subject. Suitable polymers include,but are by no means limited to, polyethylene glycol (PEG) (e.g., PEGwith a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDaor 40 kDa), dextran and monomethoxypolyethylene glycol (mPEG). Methodsfor producing PEGylated anti-IL8 antibodies which are described in U.S.Pat. No. 6,133,426 can be applied to the production of PEGylatedanti-NPC1L1 antibodies of the invention. Lee, et al., (1999) (Bioconj.Chem. 10:973-981) discloses PEG conjugated single-chain antibodies. Wen,et al., (2001) (Bioconj. Chem. 12:545-553) discloses conjugatingantibodies with PEG which is attached to a radiometal chelator(diethylenetriaminpentaacetic acid (DTPA)).

[0087] The antibody molecules of the invention may also be conjugatedwith labels such as ⁹⁹Tc, ⁹⁰Y, ¹¹¹In, ³²P, ¹⁴C, ¹²⁵I, ³H, ¹³¹I, ¹¹C,150, 13N, ¹⁸F, ³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ⁵⁹Fe, ⁵⁷Se, ¹⁵²Eu, ⁶⁷CU,²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, 234Th, ⁴⁰K, ¹⁵⁷Gd, ⁵⁵Mn, ⁵²Tr or ⁵⁶Fe.

[0088] The antibody molecules of the invention may also be conjugatedwith fluorescent or chemilluminescent labels, including fluorophoressuch as rare earth chelates, fluorescein and its derivatives, rhodamineand its derivatives, isothiocyanate, phycoerythrin, phycocyanin,allophycocyanin, o-phthaladehyde, fluorescamine, ¹⁵²Eu, dansyl,umbelliferone, luciferin, luminal label, isoluminal label, an aromaticacridinium ester label, an imidazole label, an acridimium salt label, anoxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones,biotin/avidin, spin labels and stable free radicals.

[0089] The antibody molecules may also be conjugated to a cytotoxicfactor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain,ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins and compounds (e.g., fatty acids), dianthin proteins,Phytoiacca americana proteins PAPI, PAPII, and PAP-S, momordicacharantia inhibitor, curcin, crotin, saponaria officinalis inhibitor,mitogellin, restrictocin, phenomycin, and enomycin.

[0090] Any method known in the art for conjugating the antibodymolecules of the invention to the various moieties may be employed,including those methods described by Hunter, et al., (1962) Nature144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al.,(1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. andCytochem. 30:407.

[0091] Methods for conjugating antibodies are conventional and very wellknown in the art.

Screening Assays

[0092] The invention allows the discovery of selective agonists andantagonists of NPC1L1 (e.g., SEQ ID NO: 2, 4 or 12) that may be usefulin treatment and management of a variety of medical conditions includingelevated serum cholesterol. Thus, NPC1L1 of this invention can beemployed in screening systems to identify agonists or antagonists.Essentially, these systems provide methods for bringing together NPC1L1,an appropriate, known ligand or agonist or antagonist, includingcholesterol, ezetimibe, BODIPY-ezetimibe (Altmann, et al., (2002)Biochim. Biophys. Acta 1580(1):77-93) or 4″,6″-bis[(2-fluorophenyl)carbamoyl]-beta-D-cellobiosyl derivative of11-ketotigogenin as described in DeNinno, et al., (1997) (J. Med. Chem.40(16):2547-54) (Merck; L-166,143), and a sample to be tested for thepresence of an NPC1L1 agonist or antagonist. A convenient method bywhich to evaluate whether a sample contains an NPC1L1 agonist orantagonist is to determine whether the sample contains a substance whichcompetes for binding between the known agonist or antagonist (e.g.,ezetimibe) and NPC1L1.

[0093] Ezetimibe can be prepared by a variety of methods well know tothose skilled in the art, for example such as are disclosed in U.S. Pat.Nos. 5,631,365, 5,767,115, 5,846,966, 6,207,822, U.S. Patent ApplicationPublication No. 2002/0193607 and PCT Patent Application WO 93/02048,each of which is incorporated herein by reference in its entirety.

[0094] “Sample”, “candidate compound” or “candidate substance” refers toa composition which is evaluated in a test or assay, for example, forthe ability to agonize or antagonize NPC1L1 (e.g., SEQ ID NO: 2, 4 or12) or a functional fragment thereof. The composition may smallmolecules, peptides, nucleotides, polynucleotides, subatomic particles(e.g., a particles, P particles) or antibodies.

[0095] Two basic types of screening systems can be used, alabeled-ligand binding assay (e.g., direct binding assay orscintillation proximity assay (SPA)) and a “cholesterol uptake” assay. Alabeled ligand for use in the binding assay can be obtained by labelingcholesterol or a known NPC1L1 agonist or antagonist with a measurablegroup (e.g., ¹²⁵I or ³H). Various labeled forms of cholesterol areavailable commercially or can be generated using standard techniques(e.g., Cholesterol-[1,2-³H(N)], Cholesterol-[1,2,6,7-³H(N)] orCholesterol-[7-³H(N)]; American Radiolabeled Chemicals, Inc; St. Louis,Mo.). In a preferred embodiment, ezetimibe is fluorescently labeled witha BODIPY group (Altmann, et al., (2002) Biochim. Biophys. Acta1580(1):77-93) or labeled with a detectable group such as ¹²⁵I or ³H.

[0096] Direct Biding Assay. Typically, a given amount of NPC1L1 of theinvention (e.g., SEQ ID NO: 2, 4 or 12) is contacted with increasingamounts of labeled ligand or known antagonist or agonist (discussedabove) and the amount of the bound, labeled ligand or known antagonistor agonist is measured after removing unbound, labeled ligand or knownantagonist or agonist by washing. As the amount of the labeled ligand orknown agonist or antagonist is increased, a point is eventually reachedat which all receptor binding sites are occupied or saturated. Specificreceptor binding of the labeled ligand or known agonist or antagonist isabolished by a large excess of unlabeled ligand or known agonist orantagonist.

[0097] Preferably, an assay system is used in which non-specific bindingof the labeled ligand or known antagonist or agonist to the receptor isminimal. Non-specific binding is typically less than 50%, preferablyless than 15%, and more preferably less than 10% of the total binding ofthe labeled ligand or known antagonist or agonist.

[0098] A nucleic acid encoding an NPC1L1 polypeptide of the invention(e.g., SEQ ID NO: 2, 4 or 12) can be transfected into an appropriatehost cell, whereby the receptor will become incorporated into themembrane of the cell. A membrane fraction can then be isolated from thecell and used as a source of the receptor for assay. Alternatively, thewhole cell expressing the receptor in the cell surface can be used in anassay. Preferably, specific binding of the labeled ligand or knownantagonist or agonist to an untransfected/untransformed host cell or toa membrane fraction from an untransfected/untransformed host cell willbe negligible.

[0099] In principle, a binding assay of the invention could be carriedout using a soluble NPC1L1 polypeptide of the invention, e.g., followingproduction and refolding by standard methods from an E. coli expressionsystem, and the resulting receptor-labeled ligand complex could beprecipitated, e.g., using an antibody against the receptor. Theprecipitate could then be washed and the amount of the bound, labeledligand or antagonist or agonist could be measured.

[0100] In the basic binding assay, the method for identifying an NPC1L1agonist or antagonist includes:

[0101] (a) contacting NPC1L1 (e.g., SEQ ID NO: 2 or 4 or 12) or asubsequence thereof, in the presence of a known amount of labeledcholesterol or known antagonist or agonist (e.g., labeled ezetimibe orlabeled L-166,143) with a sample to be tested for the presence of anNPC1L1 agonist or antagonist; and

[0102] (b) measuring the amount of labeled cholesterol or knownantagonist or agonist bound to the receptor.

[0103] An NPC1L1 antagonist or agonist in the sample is identified bymeasuring substantially reduced binding of the labeled cholesterol orknown antagonist or agonist to NPC1L1, compared to what would bemeasured in the absence of such an antagonist or agonist. For example,reduced binding between [³H]-cholesterol and NPC1L1 in the presence of asample might suggest that the sample contains a substance which iscompeting against [³H]-cholesterol for NPC1L1 binding.

[0104] Alternatively, a sample can be tested directly for binding toNPC1L1 (e.g., SEQ ID NO: 2, 4 or 12). A basic assay of this type mayinclude the following steps:

[0105] (a) contacting NPC1L1 (e.g., SEQ ID NO: 2 or 4 or 12) or asubsequence thereof with a labeled candidate compound (e.g.,[³H]-ezetimibe); and

[0106] (b) detecting binding between the labeled candidate compound andNPC1L1.

[0107] A candidate compound which is found to bind to NPC1L1 mayfunction as an agonist or antagonist of NPC1L1 (e.g., by inhibition ofcholesterol uptake).

[0108] SPA Assay. NPC1L1 antagonists or agonists may also be measuredusing scintillation proximity assays (SPA). SPA assays are conventionaland very well known in the art; see, for example, U.S. Pat. No.4,568,649. In SPA, the target of interest is immobilised to a smallmicrosphere approximately 5 microns in diameter. The microsphere,typically, includes a solid scintillant core which has been coated witha polyhydroxy film, which in turn contains coupling molecules, whichallow generic links for assay design. When a radioisotopically labeledmolecule binds to the microsphere, the radioisotope is brought intoclose proximity to the scintillant and effective energy transfer fromelectrons emitted by the isotope will take place resulting in theemission of light. While the radioisotope remains in free solution, itis too distant from the scintillant and the electron will dissipate theenergy into the aqueous medium and therefore remain undetected.Scintillation may be detected with a scintillation counter. In general,³H and ¹²⁵I labels are well suited to SPA.

[0109] For the assay of receptor-mediated binding events, the lectinwheat germ agglutinin (WGA) may be used as the SPA bead couplingmolecule (Amersham Biosciences; Piscataway, N.J.). The WGA coupled beadcaptures glycosylated, cellular membranes and glycoproteins and has beenused for a wide variety of receptor sources and cultured cell membranes.The receptor is immobilized onto the WGA-SPA bead and a signal isgenerated on binding of an isotopically labeled ligand. Other couplingmolecules which may be useful for receptor binding SPA assays includepoly-L-lysine and WGA/polyethyleneimine (Amersham Biosciences;Piscataway, N.J.). See, for example, Berry, J. A., et al., (1991)Cardiovascular Pharmacol. 17 (Suppl.7): S143-S145; Hoffman, R., et al.,(1992) Anal. Biochem. 203: 70-75; Kienhus, et al., (1992) J. ReceptorResearch 12: 389-399; Jing, S., et al., (1992) Neuron 9: 1067-1079.

[0110] The scintillant contained in SPA beads may include, for example,yttrium silicate (YSi), yttrium oxide (YOx), diphenyloxazole orpolyvinyltoluene (PVT) which acts as a solid solvent fordiphenylanthracine (DPA).

[0111] SPA assays may be used to analyze whether a sample is an NPC1 L1antagonist or agonist. In these assays, a host cell which expressesNPC1L1 (e.g., SEQ ID NO: 2 or 4 or 12) on the cell surface or a membranefraction thereof is incubated with SPA beads (e.g., WGA coated YOx beadsor WGA coated YSi beads) and labeled, known ligand or agonist orantagonist (e.g., ³H-cholesterol, 3-ezetimibe or ¹²⁵I-ezetimibe). Theassay mixture further includes either the sample to be tested or a blank(e.g., water). After an optional incubation, scintillation is measuredusing a scintillation counter. An NPC1L1 agonist or antagonist may beidentified in the sample by measuring substantially reducedfluorescence, compared to what would be measured in the absence of suchagonist or antagonist (blank). Measuring substantially reducedfluorescence may suggest that the sample contains a substance whichcompetes for NPC1L1 binding with the known ligand, agonist orantagonist.

[0112] Alternatively, a sample may be identified as an antagonist oragonist of NPC1L1 by directly detecting binding in a SPA assay. In thisassay, a labeled version of a candidate compound to be tested may be putin contact with the host cell expressing NPC1L1 or a membrane fractionthereof which is bound to the SPA bead. Fluorescence may then be assayedto detect the presence of a complex between the labeled candidatecompound and the host cell or membrane fraction expressing NPC1L1. Acandidate compound which binds to NPC1L1 may possess NPC1L1 agonistic orantagonistic activity.

[0113] Host cells expressing NPC1L1 may be prepared by transforming ortransfecting a nucleic acid encoding an NPC1L1 of the invention into anappropriate host cell, whereby the receptor becomes incorporated intothe membrane of the cell. A membrane fraction can then be isolated fromthe cell and used as a source of the receptor for assay. Alternatively,the whole cell expressing the receptor on the cell surface can be usedin an assay. Preferably, specific binding of the labeled ligand or knownantagonist or agonist to an untransfected/untransformed host cell ormembrane fraction from an untransfected/untransformed host cell will benegligible. Preferred host cells include Chinese Hamster Ovary (CHO)cells, murine macrophage J774 cells or any other macrophage cell lineand human intestinal epithelial Caco2 cells.

[0114] Cholesterol Uptake Assay. Assays may also be performed todetermine if a sample can agonize or antagonize NPC1L1 mediatedcholesterol uptake. In these assays, a host cell expressing NPC1L1(e.g., SEQ ID NO: 2 or 4 or 12) on the cell surface (discussed above)can be contacted with detectably labeled cholesterol (e.g.,³H-cholesterol or ¹²⁵I-cholesterol) along with either a sample or ablank. After an optional incubation, the cells can be washed to removeunabsorbed cholesterol. Cholesterol uptake can be determined bydetecting the presence of labeled cholesterol in the host cells. Forexample, assayed cells or lysates or fractions thereof (e.g., fractionsresolved by thin-layer chromatography) can be contacted with a liquidscintillant and scintillation can be measured using a scintillationcounter.

[0115] In these assays, an NPC1L1 antagonist in the sample may beidentified by measuring substantially reduced uptake of labeledcholesterol (e.g., ³H-cholesterol), compared to what would be measuredin the absence of such an antagonist and an agonist may be identified bymeasuring substantially increased uptake of labeled cholesterol (e.g.,³H-cholesterol), compared to what would be measured in the absence ofsuch an agonist.

Pharmaceutical Compositions

[0116] NPC1L1 agonists and antagonists discovered, for example, by thescreening methods described above may be used therapeutically (e.g., ina pharmaceutical composition) to stimulate or block the activity ofNPC1L1 and, thereby, to treat any medical condition caused or mediatedby the receptors. For example, the antibody molecules of the inventionmay also be used therapeutically (e.g., in a pharmaceutical composition)to bind NPC1 L1 and, thereby, block the ability of the receptor to bindcholesterol. Blocking the binding of the cholesterol may preventabsorption of the molecule (e.g., by intestinal cells such asenterocytes). Blocking absorption of cholesterol may be a useful way tolower serum cholesterol levels in a subject and, thereby, reduce theincidence of, for example, hyperlipidemia, atherosclerosis, coronaryheart disease, stroke or arteriosclerosis.

[0117] The term “subject” or “patient” includes any organism, preferablyanimals, more preferably mammals (e.g., mice, rats, rabbits, dogs,horses, primates, cats) and most preferably humans.

[0118] The term “pharmaceutical composition” refers to a compositionincluding an active ingredient and a pharmaceutically acceptable carrierand/or adjuvant.

[0119] Although the compositions of this invention could be administeredin simple solution, they are more typically used in combination withother materials such as carriers, preferably pharmaceutically acceptablecarriers. Useful, pharmaceutically acceptable carriers can be anycompatible, non-toxic substances suitable for delivering thecompositions of the invention to a subject. Sterile water, alcohol,fats, waxes, and inert solids may be included in a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable adjuvants (bufferingagents, dispersing agents) may also be incorporated into thepharmaceutical composition.

[0120] Preferably, the pharmaceutical compositions of the invention arein the form of a pill or capsule. Methods for formulating pills andcapsules are very well known in the art. For example, for oraladministration in the form of tablets or capsules, the active drugcomponent may be combined with any oral, non-toxic pharmaceuticallyacceptable inert carrier, such as lactose, starch, sucrose, cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, talc,mannitol, ethyl alcohol (liquid forms) and the like. Moreover, whendesired or needed, suitable binders, lubricants, disintegrating agentsand coloring agents may also be incorporated in the mixture. Suitablebinders include starch, gelatin, natural sugars, corn sweeteners,natural and synthetic gums such as acacia, sodium alginate,carboxymethylcellulose, polyethylene glycol and waxes. Among thelubricants there may be mentioned for use in these dosage forms, boricacid, sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrants include starch, methylcellulose, guar gum and the like.Sweetening and flavoring agents and preservatives may also be includedwhere appropriate.

[0121] The pharmaceutical compositions of the invention may beadministered in conjunction with a second pharmaceutical composition orsubstance. In preferred embodiments, the second composition includes acholesterol-lowering drug. When a combination therapy is used, bothcompositions may be formulated into a single composition forsimultaneous delivery or formulated separately into two or morecompositions (e.g., a kit).

[0122] The formulations may conveniently be presented in unit dosageform and may be prepared by any methods well known in the art ofpharmacy. See, e.g., Gilman et al. (eds.) (1990), The PharmacologicalBases of Therapeutics, 8th Ed., Pergamon Press; and Remington'sPharmaceutical Sciences, supra, Easton, Pa.; Avis et al. (eds.) (1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York;Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: TabletsDekker, New York; and Lieberman et al. (eds.) (1990), PharmaceuticalDosage Forms: Disperse Systems Dekker, New York.

[0123] The dosage regimen involved in a therapeutic application may bedetermined by a physician, considering various factors which may modifythe action of the therapeutic substance, e.g., the condition, bodyweight, sex and diet of the patient, the severity of any infection, timeof administration, and other clinical factors. Often, treatment dosagesare titrated upward from a low level to optimize safety and efficacy.Dosages may be adjusted to account for the smaller molecular sizes andpossibly decreased half-lives (clearance times) followingadministration.

[0124] An “effective amount” of an antagonist of the invention may be anamount that will detectably reduce the level of intestinal cholesterolabsorption or detectably reduce the level of serum cholesterol in asubject administered the composition.

[0125] Typical protocols for the therapeutic administration of suchsubstances are well known in the art. Pharmaceutical composition of theinvention may be administered, for example, by any parenteral ornon-parenteral route.

[0126] Pills and capsules of the invention can be administered orally.Injectable compositions can be administered with medical devices knownin the art; for example, by injection with a hypodermic needle.

[0127] Injectable pharmaceutical compositions of the invention may alsobe administered with a needleless hypodermic injection device; such asthe devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824 or 4,596,556.

Anti-Sense

[0128] The present invention also encompasses anti-senseoligonucleotides capable of specifically hybridizing to mRNA encodingNPC1L1 (e.g., any of SEQ ID NOs: 1, 3, 5-11 or 13) having an amino acidsequence defined by, for example, SEQ ID NO: 2 or 4 or 12 or asubsequence thereof so as to prevent translation of the mRNA.Additionally, this invention contemplates anti-sense oligonucleotidescapable of specifically hybridizing to the genomic DNA molecule encodingNPC1L1, for example, having an amino acid sequence defined by SEQ ID NO:2 or 4 or 12 or a subsequence thereof.

[0129] This invention further provides pharmaceutical compositionscomprising (a) an amount of an oligonucleotide effective to reduceNPC1L1-mediated cholesterol absorption by passing through a cellmembrane and binding specifically with mRNA encoding NPC1L1 in the cellso as to prevent its translation and (b) a pharmaceutically acceptablecarrier capable of passing through a cell membrane. In an embodiment,the oligonucleotide is coupled to a substance that inactivates mRNA. Inanother embodiment, the substance that inactivates mRNA is a ribozyme.

EXAMPLES

[0130] The following examples are provided to more clearly describe thepresent invention and should not be construed to limit the scope of theinvention in any way.

Example 1 Cloning and Expression of Rat, Mouse and Human NPC1L1

[0131] Rat NPC, mouse NPC1L1 or human NPC1L1 can all conveniently beamplified using polymerase chain reaction (PCR). In this approach, DNAfrom a rat, mouse or human cDNA library can be amplified usingappropriate primers and standard PCR conditions. Design of primers andoptimal amplification conditions constitute standard techniques whichare commonly known in the art.

[0132] An amplified NPC1L1 gene may conveniently be expressed, again,using methods which are commonly known in the art. For example, NPC1L1may be inserted into a pET-based plasmid vector (Stratagene; La Joola,Calif.), downstream of the T7 RNA polymerase promoter. The plasmid maythen be transformed into a T7 expression system (e.g., BL21DE3 E. colicells), grown in a liquid culture and induced (e.g., by adding IPTG tothe bacterial culture).

Example 2 Direct Binding Assay

[0133] Membrane preparation: Caco2 cells transfected with an expressionvector containing a polynucleotide encoding NPC1L1 (e.g., SEQ ID NO: 2,4 or 12) are harvested by incubating in 5 mM EDTA/phosphate-bufferedsaline followed by repeated pipeting. The cells are centrifuged 5 min at1000×g. The EDTA/PBS is decanted and an equal volume of ice-cold 50 mMTris-HCl, pH 7.5 is added and cells are broken up with a Polytron (PT10tip, setting 5, 30 sec). Nuclei and unbroken cells are sedimented at1000×g for 10 min and then the supernatant is centrifuged at 50,000×gfor 10 min. The supernatant is decanted, the pellet is resuspended byPolytron, a sample is taken for protein assay (bicinchoninic acid,Pierce), and the tissue is again centrifuged at 50,000×g. Pellets arestored frozen at −20° C.

[0134] Binding assay: For saturation binding, four concentrations of[³H]-ezetimibe (15 Ci/mmol) are incubated without and with 10⁻⁵ Mezetimibe in triplicate with 50 μg of membrane protein in a total volumeof 200 μl of 50 mM Tris-HCl, pH 7.5, for 30 min at 30° C. Samples arefiltered on GF/B filters and washed three times with 2 ml of cold Trisbuffer. Filters are dried in a microwave oven, impregnated with Meltilexwax scintillant, and counted at 45% efficiency. For competition bindingassays, five concentrations of a sample are incubated in triplicate with18 nM [³H]-ezetimibe and 70 μg of membrane protein under the conditionsdescribed above. Curves are fit to the data with Prism (GraphPadSoftware) nonlinear least-squares curve-fitting program and K_(i) valuesare derived from IC₅₀ values according to Cheng and Prusoff (Cheng, Y.C., et al., (1973) Biochem. Pharmacol. 22:3099-3108).

Example 3 SPA Assay

[0135] For each well of a 96 well plate, a reaction mixture of 10 μghuman, mouse or rat NPC1L1-CHO overexpressing membranes (Biosignal) and200 μg/well YSi-WGA-SPA beads (Amersham) in 100 μl is prepared in NPC1L1assay buffer (25 mM HEPES, pH 7.8, 2 mM CaCl₂, 1 mM MgCl₂, 125 mM NaCl,0.1% BSA). A 0.4 nM stock ofligand-[¹²⁵]-ezetimibe- is prepared in theNPC1L1 assay buffer. The above solutions are added to a 96-well assayplate as follows: 50 μl NPC1L1 assay buffer, 100 [1 of reaction mixture,50 μl of ligand stock (final ligand concentration is 0.1 nM). The assayplates are shaken for 5 minutes on a plate shaker, then incubated for 8hours before cpm/well are determined in Microbeta Trilux counter(PerkinElmer).

[0136] These assays will indicate that [¹²⁵I]-ezetimibe binds to thecell membranes expressing human, mouse or rat NPC1L1. Similar resultswill be obtained if the same experiment is performed with radiolabeledcholesterol (e.g., ¹²⁵I-cholesterol).

Example 4 Cholesterol Uptake Assay

[0137] CHO cells expressing either SR-B1 or three different clones ofrat NPC1L1 or one clone of mouse NPC1L1 were starved overnight incholesterol free media then dosed with [³H]-cholesterol in a mixedsynthetic micelle emulsion for 4 min, 8 min, 12 min or 24 min in theabsence or presence of 10 μM ezetimibe. The cells were harvested and thelipids were organically extracted. The extracted lipids were spotted onthin-layer chromatography (TLC) plates and resolved within an organicvapor phase. The free cholesterol bands for each assay were isolated andcounted in a scintillation counter.

[0138] The SR-B1 expressing cells exhibited an increase in[³H]-cholesterol uptake as early as 4 min which was also inhibited byezetimibe. The three rat clones and the one mouse clone appeared to givebackground levels of [³H]-cholesterol uptake which was similar to thatof the untransformed CHO cell.

[0139] These experiments will yield data demonstrating that CHO cellscan perform mouse, rat and human NPC1L1-dependent uptake of[³H]-cholesterol when more optimal experimental conditions aredeveloped.

Example 5 Expression of Rat NPC1L1 in Wistar Rat Tissue

[0140] In these experiments, the expression of rat NPC1L1 mRNA, inseveral rat tissues, was evaluated. The tissues evaluated wereesophagus, stomach, duodenum, jejunum, ileum, proximal colon, distalcolon, liver, pancreas, heart, aorta, spleen, lung, kidney, brain,muscle, testes, ovary, uterus, adrenal gland and thyroid gland. TotalRNA samples were isolated from at least 3 male and 3 female animals andpooled. The samples were then subjected to real time quantitative PCRusing Taqman analysis using standard dual-labeled fluorogenicoligonucleotide probes. Typical probe design incorporated a 5′ reporterdye (e.g., 6FAM (6-carboxyfluorescein) or VIC) and a 3′ quenching dye(e.g., TAMRA (6-carboxytetramethyl-rhodamine)). rat NPC1L1: (SEQ IDNO:14) Forward: TCTTCACCCTTGCTCTTTGC (SEQ ID NO:15) Reverse:AATGATGGAGAGTAGGTTGAGGAT (SEQ ID NO:16) Probe:[6FAM]TGCCCACCTTTGTTGTCTGCTACC[TAMRA] ratβ-actin: (SEQ ID NO:17)Forward: ATCGCTGACAGGATGCAGAAG (SEQ ID NO:18) Reverse:TCAGGAGGAGCAATGATCTTGA (SEQ ID NO:19) Probe:[VIC]AGATTACTGCCCTGGCTCCTAGCACCAT[TAMRA]

[0141] PCR reactions were run in 96-well format with 25 μl reactionmixture in each well containing: Platinum SuperMix (12.5 μl), ROXReference Dye (0.5 ul), 50 mM magnesium chloride (2 μl), cDNA from RTreaction (0.2 μl). Multiplex reactions contained gene specific primersat 200 nM each and FAM labeled probe at 100 nM and gene specific primersat 100 nM each and VIC labeled probe at 50 nM. Reactions were run with astandard 2-step cycling program, 95° C. for 15 sec and 60° C. for 1 min,for 40 cycles.

[0142] The highest levels of expression were observed in the duodenum,jejunum and ileum tissue. These data indicate that NPC1L1 plays a rolein cholesterol absorption in the intestine.

Example 6 Expression of Mouse NPC1L1 in Mouse Tissue

[0143] In these experiments, the expression of mouse NPC1L1 mRNA, inseveral tissues, was evaluated. The tissues evaluated were adrenalgland, BM, brain, heart, islets of langerhans, LI, small intestine,kidney, liver, lung, MLN, PLN, muscle, ovary, pituitary gland, placenta,Peyers Patch, skin, spleen, stomach, testes, thymus, thyroid gland,uterus and trachea. Total RNA samples were isolate from at least 3 maleand 3 female animals and pooled. The samples were then subjected to realtime quantitative PCR using Taqman analysis using the following primersand probes: mouse NPC1L1: Forward: ATCCTCATCCTGGGCTTTGC (SEQ ID NO:20)Reverse: GCAAGGTGATCAGGAGGTTGA (SEQ ID NO:21) Probe:[6FAM]CCCAGCTTATCCAGATTTTCTTCTTCCGC[TAMRA] (SEQ ID NO:22)

[0144] The highest levels of expression were observed in the Peyer'sPatch, small intestine, gall bladder and stomach tissue. These data areconsistent with a cholesterol absorption role for NPC1L1 which takesplace in the digestive system.

Example 7 Expression of Human NPC1L1 in Human Tissue

[0145] In these experiments, the expression level of human NPC1L1 mRNAwas evaluated in 2045 samples representing 46 normal tissues.Microarray-based gene expression analysis was performed on theAffymetrix HG-U95 GeneChip using a cRNA probe corresponding to basepairs 4192-5117 (SEQ ID NO: 43) in strict accordance to Affymetrix'sestablished protocols. Gene Chips were scanned under low photomultiplier tube (PMT), and data were normalized using either AffymetrixMAS 4.0 or MAS 5.0 algorithms. In addition “spike ins” for most sampleswere used to construct a standard curve and obtain RNA concentrationvalues according Gene Logic algorithms and procedures. A summary ofthese results are indicated, below, in Table 2. TABLE 2 Expression levelf NPC1L1 mRNA in various human tissues.

[0146] Shaded data corresponds to tissues wherein the highest levels ofNPC1L1 mRNA was detected. The “Present” column indicates the proportionof specified tissue samples evaluated wherein NPC1L1 mRNA was detected.The “Absent” column indicates the proportion of specified tissue samplesevaluated wherein NPC1L1 RNA was not detected.

[0147] The “lower 25%”, “median” and “upper 75%” columns indicatestatistical distribution of the relative NPC1L1 signal intensitiesobserved for each set of tissue evaluated.

Example 8 Distribution of Rat NPC1L1, Rat IBA T or Rat SR-B1 mRNA in RatSmall Intestine

[0148] In these experiments, the distribution of rat NPC1L1 mRNA alongthe proximal-distal axis of rat small intestines was evaluated.Intestines were isolated from five independent animals and divided into10 sections of approximately equal length. Total RNA was isolated andanalyzed, by real time quantitative PCR using Taqman analysis, forlocalized expression levels of rat NPC1L1, rat IBAT (ileal bile acidtransporter) or rat SR-B1 mRNA. The primers and probes used in theanalysis were: rat NPC1L1: Forward: TCTTCACCCTTGCTCTTTGC (SEQ ID NO:23)Reverse: AATGATGGAGAGTAGGTTGAGGAT (SEQ ID NO:24) Probe:[6FAM]TGCCCACCTTTGTTGTCTGCTACC[TAMRA] (SEQ ID NO:25) rat Villin:Forward: AGCACCTGTCCACTGAAGATTTC (SEQ ID NO:26) Reverse:TGGACGCTGAGCTTCAGTTCT (SEQ ID NO:27) Probe:[VIC]CTTCTCTGCGCTGCCTCGATGGAA[TAMRA] (SEQ ID NO:28) rat SR-BI: Forward:AGTAAAAAGGGCTCGCAGGAT (SEQ ID NO:29) Reverse: GGCAGCTGGTGACATCAGAGA (SEQID NO:30) Probe: [6FAM]AGGAGGCCATGCAGGCCTACTCTGA[TAMRA] (SEQ ID NO:31)rat IBAT: Forward: GAGTCCACGGTCAGTCCATGT (SEQ ID NO:32) Reverse:TTATGAACAACAATGCCAAGCAA (SEQ ID NO:33) Probe:[6FAM]AGTCCTTAGGTAGTGGCTTAGTCCCTGGAAGCTC[TAMRA] (SEQ ID NO:34)

[0149] The mRNA expression levels of each animal intestinal section wereanalyzed separately, then the observed expression level was normalizedto the observed level of villin mRNA in that intestinal section. Theobserved, normalized mRNA expression levels for each section where thenaveraged.

[0150] The expression level of NPC1L1 and SR-B1 were highest in thejejunum (sections 2-5) as compared to that of the more distal ileumsections. Since the jejunum is believed to be the site of cholesterolabsorption, these data suggest such a role for rat NPC1L1. IBATdistribution favoring the ileum is well document and served as a controlfor the experiment.

Example 9 In Situ Analysis of Rat NPC1L1 mRNA in Rat Jejunum Tissue

[0151] The localization of rat NPC1L1 mRNA was characterized by in situhybridization analysis of rat jejunum serial sections. The probes usedin this analysis were: T7-sense probe:GTAATACGACTCACTATAGGGCCCTGACGGTCCTTCCTGA (SEQ ID NO:35) GGGAATCTTCACT7-antisense probe: GTAATACGACTCACTATAGGGCCTGGGAAGTTGGTCAT (SEQ IDNO:36) GGCCACTCCAGC

[0152] The RNA probes were synthesized using T7 RNA polymeraseamplification of a PCR amplified DNA fragment corresponding rat NPC1L1nucleotides 3318 to 3672 (SEQ ID NO 1). Sense and anti-sensedigoxigenin-UTP labeled cRNA probes were generated from the T7 promoterusing the DIG RNA Labeling Kit following the manufacturer'sinstructions. Serial cryosections rat jejunum were hybridized with thesense and antiisense probes. Digoxigenin labeling was detected with theDIG Nucleic Acid Detection Kit based on previous methods. A positivesignal is characterized by the deposition of a red reaction product atthe site of hybridization.

[0153] The anti-sense probe showed strong staining of epithelium alongthe crypt-villus axis under low magnification (40×). The observed ratNPC1L1 mRNA expression levels may have been somewhat greater in thecrypts than in the villus tips. Under high magnification (200×),staining was observed in the enterocytes but not in the goblet cells. Alack of staining observed with the sense probe (control) confirmed thehigh specificity of the NPC1L1 anti-sense signal. These data providedfurther evidence of the role of rat NPC1L1 in intestinal cholesterolabsorption.

Example 10 FACS Analysis of Fluorescently Labeled Ezetimibe Binding toTransiently Transfected CHO Cells

[0154] In these experiments, the ability of BODIPY-labeled ezetimibe(Altmann, et al., (2002) Biochim. Biophys. Acta 1580(1):77-93) to bindto NPC1L1 and SR-B1 was evaluated. “BODIPY” is a fluorescent group whichwas used to detect the BODIPY-ezetimibe. Chinese hamster ovary (CHO)cells were transiently transfected with rat NPC1L1 DNA (rNPC1L1/CHO),mouse NPC1L1 DNA (mNPC1L1/CHO), mouse SR-B1 DNA (mSRB1/CHO) or EGFP DNA(EGFP/CHO). EGFP is enhanced green fluorescent protein which was used asa positive control. The transfected CHO cells or untransfected CHO cellswere then stained with 100 nM BODIPY-labeled ezetimibe and analyzed byFACS. Control experiments were also performed wherein the cells were notlabeled with the BODIPY-ezetimibe and wherein untransfected CHO cellswere labeled with the BODIPY-ezetimibe.

[0155] No staining was observed in the untransfected CHO, rNPC1L1/CHO ormNPC1L1/CHO cells. Fluorescence was detected in the positive-controlEGFP/CHO cells. Staining was also detected in the mouse SR-B1/CHO cells.These data show that, under the conditions tested, BODIPY-ezetimibe iscapable of binding to SR-B1 and that such binding is not ablated by thepresence of the fluorescent BODIPY group. When more optimal conditionsare determined, BODIPY-ezetimibe will be shown to label the rNPC1L1/CHOand mNPC1L1/CHO cells.

Example 11 FACS Analysis of Transiently Transfected CHO Cells Labeledwith Anti-FLAG Antibody M2

[0156] In these experiments, the expression of FLAG-tagged NPC1L1 on CHOcells was evaluated. CHO cells were transiently transfected with mouseNPC1L1 DNA, rat NPC1L1 DNA, FLAG-rat NPC1L1 DNA or FLAG-mouse NPC1L1DNA. The 8 amino acid FLAG tag used was DYKDDDDK (SEQ ID NO: 37) whichwas inserted on the amino-terminal extracellular loop just past thesecretion signal sequence. The cells were incubated with commerciallyavailable anti-FLAG monoclonal mouse antibody M2 followed by aBODIPY-tagged anti-mouse secondary antibody. The treated cells were thenanalyzed by FACS.

[0157] The M2 antibody stained the CHO cells transfected with FLAG-ratNPC1L1 DNA and with FLAG-mouse NPC1L1. No staining was observed in theCHO cells transfected with mouse NPC1L1 DNA and with rat NPC1L1 DNA.These data showed that rat NPC1L1 and mouse NPC1L1 possess nosignificant, inherent fluorescence and are not bound by the anti-FLAGantibody. The observed, FLAG-dependent labeling of the cells indicatedthat the FLAG-mouse NPC1L1 and FLAG-rat NPC1L1 proteins are localized atthe cell membrane of the CHO cells.

Example 12 FACS Analysis of FLAG-Rat NPC1L1-EGFP Chimera in TransientlyTransfected CHO Cells

[0158] In these experiments, the surface and cytoplasmic localization ofrat NPC1L1 in CHO cells was evaluated. CHO cells were transientlytransfected with FLAG-rat NPC1L1 DNA or with FLAG-rat NPC1L1-EGFP DNA.In these fusions, the FLAG tag is at amino-terminus of rat NPC1L1 andEGFP fusion is at the carboxy-terminus of rat NPC1L1. The cells werethen stained with the M2 anti-FLAG mouse (primary) antibody followed bysecondary staining with a BODIPY-labeled anti-mouse antibody. In controlexperiments, cells were stained with only the secondary antibody and notwith the primary antibody (M2). The stained cells were then analyzed byFACS.

[0159] In a control experiment, FLAG-rat NPC1L1 transfected cells werestained with BODIPY anti-mouse secondary antibody but not with theprimary antibody. The data demonstrated that the secondary, anti-mouseantibody possessed no significant specificity for FLAG-rat NPC1L1 andthat the FLAG-rat NPC1L1, itself, possesses no significant fluorescence.

[0160] In another control experiment, unlabeled FLAG-rat NPC1L1-EGFPcells were FACS analyzed. In these experiments, autofluorescence of theenhanced green fluorescent protein (EGFP) was detected.

[0161] FLAG-rat NPC1L1 cells were stained with anti-FLAG mouse antibodyM2 and with the BODIPY-labeled anti-mouse secondary antibody and FACSanalyzed. The data from this analysis showed that the cells were labeledwith the secondary, BODIPY-labeled antibody which indicated expressionof the FLAG-rat NPC1L1 protein on the surface of the CHO cells.

[0162] FLAG-rat NPC1L1-EGFP cells were stained with anti-FLAG mouseantibody M2 and with the BODIPY-labeled anti-mouse secondary antibodyand FACS analyzed. The data from this analysis showed that both markers(BODIPY and EGFP) were present indicating surface expression of thechimeric protein. The data also indicated that a portion of the proteinwas located within the cells and may be associated with transportvesicles. These data supported a role for rat NPC1L1 in vesiculartransport of cholesterol or protein expressed in subcellular organellessuch as the rough endoplasmic reticulum.

Example 13 FACS Analysis and Fluorescent Microscopy of FLAG-ratNPC1L1-EGFP Chimera in a Cloned CHO Cell Line

[0163] In these experiments, the cellular localization of rat NPC1L1 wasevaluated by FACS analysis and by immunohistochemistry. CHO cells weretransfected with FLAG-rat NPC1L1-EGFP DNA and stained with anti-FLAGmouse antibody M2 and then with a BODIPY-labeled anti-mouse secondaryantibody. In the fusion, the FLAG tag is at the amino-terminus of ratNPC1L1 and the enhanced green fluorescent protein (EGFP) tag is locatedat the carboxy-terminus of the rat NPC1L1. The stained cells were thenanalyzed by FACS and by fluorescence microscopy.

[0164] Cells transfected with FLAG-rat NPC1L1-EGFP DNA were stained withthe anti-FLAG mouse antibody M2 and then with the BODIPY-labeledanti-mouse secondary antibody. FACS analysis of the cells detected bothmarkers indicating surface expression of the chimeric protein.

[0165] FLAG-rat NPC1L1-EGFP transfected cells were analyzed byfluorescent microscopy at 63× magnification. Fluorescent microscopicanalysis of the cells indicated non-nuclear staining with significantperinuclear organelle staining. Resolution of the image could notconfirm the presence of vesicular associated protein. These dataindicated that the fusion protein was expressed on the cell membrane ofCHO cells.

Example 14 Generation of Polyclonal Anti-Rat NPC1L1 Rabbit Antibodies

[0166] Synthetic peptides (SEQ ID NO: 39-42) containing an amino- orcarboxy-terminal cysteine residue were coupled to keyhole limpethemocyanin (KLH) carrier protein through a disulfide linkage and used asantigen to raise polyclonal antiserum in New Zealand white rabbits(range 3-9 months in age). The KLH-peptide was emulsified by mixing withan equal volume of Freund's Adjuvant, and injected into threesubcutaneous dorsal sites. Prior to the 16 week immunization schedule apre-immune sera sample was collected which was followed by a primaryinjection of 0.25 mg KLH-peptide and 3 scheduled booster injections of0.1 mg KLH-peptide. Animals were bled from the auricular artery and theblood was allowed to clot and the serum was then collected bycentrifugation

[0167] The anti-peptide antibody titer was determined with an enzymelinked immunosorbent assay (ELISA) with free peptide bound in solidphase (1 μg/well). Results are expressed as the reciprocal of the serumdilution that resulted in an OD₄₅₀ of 0.2. Detection was obtained usingthe biotinylated anti-rabbit IgG, horse radish peroxidase streptavidin(HRP-SA) conjugate, and ABTS.

Example 15 FACS Analysis of Rat NPC1L1 Expression in CHO CellsTransiently Transfected with Rat NPC1L1 DNA Using Rabbit Anti-rat NPC1L1Antisera

[0168] In these experiments, the expression of rat NPC1L1 on the surfaceof CHO cells was evaluated. CHO cells were transfected with rat NPC1L1DNA, then incubated with either rabbit preimmune serum or with 10 weekanti-rat NPC1L1 serum described, above, in Example 14 (i.e., A0715,A0716, A0867 or A0868). Cells labeled with primary antisera were thenstained with a BODIPY-modified anti-rabbit secondary antibody followedby FACS analysis.

[0169] No antibody surface labeling was observed for any of thepre-immune sera samples. Specific cell surface labeling of rat NPC1L1transfected cells was observed for both A0715 and A0868. Antisera A0716and A0867 did not recognize rat NPC1L1 surface expression in this assayformat. This indicates that the native, unfused rat NPC1L1 protein isexpressed in the CHO cells and localized to the CHO cell membranes. Cellsurface expression of NPC1L1 is consistent with a role in intestinalcholesterol absorption.

Example 16 FACS Analysis of CHO Cells Transiently Transfected withFLAG-Mouse NPC1L1 DNA or FLAG-Rat NPC1L1 DNA or Untransfected CHO CellsUsing Rabbit Anti-rat NPC1L1 Antisera

[0170] In these experiments, the expression of FLAG-mouse NPC1L1 andFLAG-rat NPC1L1 in CHO cells was evaluated. CHO cells were transientlytransfected with FLAG-mouse NPC1L1 DNA or with FLAG-rat NPC1L1 DNA. TheFLAG-mouse NPC1L1 and FLAG-rat NPC1L1 transfected cells were labeledwith either A0801, A0802, A0715 or A0868 sera (see Example 14) or withanti-FLAG antibody, M2. The labeled cells were then stained withBODIPY-labeled anti-rabbit secondary antibody and FACS analyzed. Theuntransfected CHO cells were analyzed in the same manner as thetransfected cell lines.

[0171] Positive staining of the untransfected CHO cells was not observedfor any of the antisera tested. Serum A0801-dependent labeling ofFLAG-rat NPC1L1 transfected cells was observed but such labeling ofFLAG-mouse NPC1L1 transfected cells was not observed. SerumA0802-dependent labeling of FLAG-mouse NPC1L1 or FLAG-rat NPC1L1transfected cells was not observed. Strong serum A0715-dependentlabeling of FLAG-rat NPC1L1 transfected cells was observed and weakserum A0715-dependent labeling of FLAG-mouse NPC1L1 transfected cellswas observed. Weak serum A0868-dependent labeling of rat NPC1L1 andmouse NPC1L1 transfected cells was observed. Strong Anti-FLAG M2antibody-dependent labeling of FLAG-rat NPC1L1 and FLAG-mouse NPC1L1transfected cells was observed. The strong M2 staining is likely to bedue to the fact that M2 is an affinity-purified, monoclonal antibody ofknown concentration. In contrast, the respective antisera arepolyclonal, unpurified and contain an uncertain concentration ofanti-rat NPC1L1 antibody. These date provide further evidence that theFLAG-mouse NPC1L1 and FLAG-rat NPC1L1 proteins are expressed in CHOcells and localized to the CHO cell membranes. Cell surface expressionof NPC1L1 is consistent with a role in intestinal cholesterolabsorption.

Example 17 Immunohistochemical Analysis of Rat Jejunum Tissue withRabbit Anti-rat NPC1L1 Antisera A0715

[0172] In these experiments, the localization of rat NPC1L1 in ratjejunum was analyzed by immunohistochemistry. Rat jejunum was removed,immediately embedded in O.C.T. compound and frozen in liquid nitrogen.Sections (6 μm) were cut with a cryostat microtome and mounted on glassslides. Sections were air dried at room temperature and then fixed inBouin's fixative. Streptavidin-biotin-peroxidase immunostaining wascarried out using Histostain-SP kit. Endogenous tissue peroxidaseactivity was blocked with a 10 minute incubation in 3% H₂O₂ in methanol,and nonspecific antibody binding was minimized by a 45 minute incubationin 10% nonimmune rabbit serum. Sections were incubated with a rabbitanti-rat NPC1L1 antisera A0715 or A0868 at a 1:500 dilution at 4° C.,followed by incubation with biotinylated goat anti-rabbit IgG and withstreptavidin-peroxidase. Subsequently, the sections were developed in anaminoethyl carbazole (AEC)-H₂O₂ staining system and counterstained withhematoxylin and examined by microscopy. A positive reaction using thisprotocol is characterized by the deposition of a red reaction product atthe site of the antigen-antibody reaction. Nuclei appeared blue from thehematoxylin counterstain. Controls were performed simultaneously on theneighboring sections from the same tissue block. Control proceduresconsisted of the following: (1) substitute the primary antibody with thepre-immune serum, (2) substitute the primary antibody with thenon-immune rabbit serum, (3) substitute the primary antibody with PBS,(4) substitute the second antibody with PBS.

[0173] The example shows tissue stained with anti-rat NPC1L1 sera A0715or with the preimmune sera analyzed at low magnification (40×) and athigh magnification (200×). The A0715-stained tissue, at lowmagnification, showed positive, strong staining of the villi epitheliallayer (enterocytes). The A0715-stained tissue at high magnificationshowed positive, strong staining of the enterocyte apical membranes. Nostaining was observed in tissue treated only with preimmune sera.Similar results were obtained with sera A0868. These data indicate thatrat NPC1L1 is expressed in rat jejunum which is consistent with a rolein intestinal cholesterol absorption.

Example 18 Labeled Cholesterol Uptake Assay

[0174] In this example, the ability of CHO cells stably transfected withrat NPC1L1 or mouse SR-B1 to take up labeled cholesterol was evaluated.In these assays, cholesterol uptake, at a single concentration, wasevaluated in a pulse-chase experiment. The data generated in theseexperiments are set forth, below, in Table 3.

[0175] Cells:

[0176] A. CHO cells stably transfected with rat NPC1L1 cDNA

[0177] B. CHO background (no transfection)

[0178] Cells were seeded at 500,000 cells/well (mL) in 12-well plates.

[0179] Procedure:

[0180] All reagents and culture plates were maintained at 37° C. unlessotherwise noted.

[0181] Starve. The maintenance media (F12 HAMS, 1% Pen/Strep, 10% FCS)was removed and the cells were rinsed with serum-free HAMS media. Theserum-free media was then replaced with 1 mL “starve” media (F12 HAMS,Pen/Strep, 5% lipoprotein deficient serum (LPDS).

[0182] One plate of each cell line was starved overnight. The remaining2 plates were designated “No Starve” (see below).

[0183] Pre-Incubation. Media was removed from all plates, rinsed withserum-free HAMS and replaced with starve media for 30 minutes.

[0184]³H-Cholesterol Pulse. The following was added directly to eachwell.

[0185] 0.5 μCi ³H-cholesterol (˜1.1×10⁶ dpm/well) in 50 μl of a mixedbile salt micelle.

[0186] 4.8 mM sodium taurocholate (2.581 mg/mL)

[0187] 0.6 mM sodium oleate (0.183 mg/mL)

[0188] 0.25 mM cholesterol (0.1 mg/mL)

[0189] Dispersed in “starve” media by ultrasonic vibration

[0190] Final media cholesterol concentration=5 μg/mL

[0191] Labeled cholesterol pulse time points were 0, 4, 12 and 24minutes. Triplicate wells for each treatment were prepared.

[0192] Wash. At the designated times, media was aspirated and the cellswere washed once with Hobbs Buffer A (50 mM Tris, 0.9% NaCl, 0.2% BSA,pH 7.4) and once with Hobbs Buffer B (50 mM Tris, 0.9% NaCl, pH 7.4 (noBSA)) at 37° C.

[0193] Processing/Analysis. Cells were digested overnight with 0.2NNaOH, 2 mL/well at room temperature. One 1.5 mL aliquot was removed fromeach well, neutralized & counted for radioactivity by scintillationcounting. Two additional 50 μl aliquots from all wells are assayed fortotal protein by the Pierce micro BCA method. The quantity of labeledcholesterol observed in the cells was normalized by the quantity ofprotein in the cells. TABLE 3 Uptake of ³H-cholesterol by CHO cellstransfected with rat NPC1L1 or mouse SR-B1 or untransfected CHO cells.Total Cholesterol, Total Cholesterol, dpm protein ±sem dpm/mg protein±sem Time, min NPC1L1 CHO NPC1L1 CHO After ³H-Cholesterol No Starve 02067 ±46 4568 ±1937 10754 ±166 22881 ±9230 4 2619 ±130 2868 ±193 15366±938 15636 ±1471 12 2868 ±193 4459 ±170 15636 ±1471 24622 ±966 24 7010±89 7204 ±173 41129 ±685 39361 ±1207 Starve 0 1937 ±273 2440 ±299 10909±1847 12429 ±1673 4 3023 ±308 2759 ±105 17278 ±1650 14307 ±781 12 2759±105 4857 ±186 14307 ±781 26270 ±1473 24 6966 ±72 7344 ±65 39196 ±17438381 ±161

Example 19 Effect of Ezetimibe on Cholesterol Uptake

[0194] The effect of ezetimibe on the ability of CHO cells stablytransfected with mouse or rat NPC1L1 or mouse SR-B1 to take up³H-labeled cholesterol was evaluated in pulse-chase experiments. OnecDNA clone of mouse NPC1L1 (C7) and three clones of rat NPC1L1 (C7, C17and C21) were evaluated. The ability of CHO cells stably transfectedwith mouse SR-B1, mouse NPC1L1 and rat NPC1L1 to take up labeledcholesterol, in the absence of ezetimibe, was also evaluated in thepulse-chase experiments. Data generated in these experiments are setforth, below, in Tables 4 and 5. Additionally, the quantity of totalcholesterol taken up by transfected and untransfected CHO cells in thepresence of four different unlabeled cholesterol concentrations was alsoevaluated. The data from these experiments is set forth, below, in Table6.

[0195] Cells:

[0196] A. CHO cells stably transfected with rat or mouse NPC1L1 cDNA

[0197] B. CHO background (no transfection)

[0198] C. SR-B] transfected CHO cells

[0199] Cells seeded at 500,000 cells/well (mL) in 12-well plates.

[0200] Procedure:

[0201] All reagents and culture plates were maintained at 37° C. unlessotherwise noted.

[0202] Starve. The maintenance media (F12 HAMS, 1% Pen/Strep, 10% FCS)was removed and the cells were rinsed with serum-free HAMS media. Theserum-free media was then replaced with 1 mL “starve” media (F12 HAMS,Pen/Strep, 5% lipoprotein deficient serum (LPDS). The cells were thenstarved overnight.

[0203] Pre-Incubation/pre-dose. Media was removed from all plates andreplaced with fresh starve media and preincubated for 30 minutes. Halfof the wells received media containing ezetimibe (stock soln in EtOH;final conc.=10 μM).

[0204]³H-Cholesterol Pulse. The following was added directly to eachwell:

[0205] 0.5 μCi ³H-cholesterol (1.1×10⁶ dpr/well) in 50 μl of a mixedbile salt micelle

[0206] 4.8 mM sodium taurocholate (2.581 mg/mL)

[0207] 0.6 mM sodium oleate (0.183 mg/mL)

[0208] 0.25 mM cholesterol (0.1 mg/mL)

[0209] Dispersed in “starve” media by ultrasonic vibration

[0210] Final media cholesterol concentration=5 μg/mL

[0211] Labeled cholesterol pulse time points were 4, 12, 24 minutes and4 hours. Triplicate wells were prepared for each treatment.

[0212] Wash. At designated times, media was aspirated and cells werewashed once with Hobbs Buffer A (50 mM Tris, 0.9% NaCl, 0.2% bovineserum albumin (BSA), pH 7.4) and once with Hobbs Buffer B (50 mM Tris,0.9% NaCl, pH 7.4 (no BSA)) at 37° C.

[0213] Processing/Analysis.

[0214] A. 4, 12, 24 minute time points: Cells were digested overnightwith 0.2N NaOH, 2 mL/well, room temperature. One 1.5 mL aliquot wasremoved from each well, neutralized & counted for radioactivity byscintillation counting.

[0215] B. 4 hour time point: The digested cells were analyzed bythin-layer chromatography to determine the content of cholesterol esterin the cells.

[0216] Extracts were spotted onto TLC plates and run for 30 minutes in 2ml hexane:isopropanol (3:2) mobile phase for 30 minutes, followed by asecond run in 1 ml hexane:isopropanol (3:2) mobile phase for 15 minutes.

[0217] C. Protein determination of cell extracts. Plates containing asample of the cell extracts were placed on orbital shaker at 120 rpm forindicated times and then extracts are pooled into 12×75 tubes. Plateswere dried and NaOH (2 ml/well) added. The protein content of thesamples were then determined. Two additional 501 μl aliquots from allwells were assayed for total protein by the Pierce micro BCA method. Thequantity of labeled cholesterol observed in the cells was normalized tothe quantity of protein in the cells. TABLE 4 Total Cholesterol inTransfected CHO Cells in the Presence and Absence of Ezetimibe. TotalCholesterol, Total Cholesterol, dpm ±sem dpm/mg protein ±sem Vehicle EZ(10 μm) Vehicle EZ (10 μm) Clones: 4 Min Pulse CHO Control 3413 ±4173222 ±26 33443 ±4070 31881 ±483 SR-B1 14207 ±51 10968 ±821 118242 ±126192474 ±2902 mNPC1L1(C7) 4043 ±419 4569 ±222 30169 ±3242 30916 ±1137rNPC1L1(C21) 3283 ±288 3769 ±147 23728 ±2111 27098 ±689 rNPC1L1(C17)3188 ±232 3676 ±134 24000 ±832 28675 ±527 rNPC1L1(C7) 1825 ±806 3268±121 15069 ±6794 27285 ±968 12 Min Pulse CHO Control 4710 ±246 4532 ±16544208 ±2702 43391 ±1197 SR-B1 16970 ±763 12349 ±298 140105 ±6523 98956±4447 mNPC1L1(C7) 6316 ±85 6120 ±755 45133 ±342 41712 ±4054 rNPC1L1(C21)5340 ±12 4703 ±231 40018 ±1181 33985 ±1928 rNPC1L1(C17) 4831 ±431 4579±257 37378 ±3461 34063 ±1619 rNPC1L1(C7) 4726 ±272 4664 ±63 39100 ±235038581 ±784 24 Min Pulse CHO Control 7367 ±232 6678 ±215 65843 ±128161764 ±2131 SR-B1 39166 ±2152 23558 ±1310 324126 ±11848 198725 ±11713mNPC1L1(C7) 10616 ±121 9749 ±482 77222 ±1040 74041 ±3670 rNPC1L1(C21)9940 ±587 8760 ±293 76356 ±9618 66165 ±2181 rNPC1L1(C17) 8728 ±721 8192±237 70509 ±5189 62279 ±4352 rNPC1L1(C7) 8537 ±148 7829 ±204 72134 ±130563482 ±368

[0218] TABLE 5 Cholesterol Ester in CHO cells in the Presence or Absenceof Ezetimibe. Cholesteryl Ester, dpm ±sem Cholesteryl Ester, dpm/mgprotein ±sem Vehicle EZ (10 μM) Vehicle EZ (10 μM) Clones: 4 Hour PulseCHO Control 652 ±13 208 ±9 5647 ±55 1902 ±87 SR-B1 47608 ±1292 9305 ±401391067 ±14391 72782 ±3181 mNPC1L1(C7) 732 ±127 453 ±118 4994 ±827 3057±776 rNPC1L1(C21) 2667 ±90 454 ±33 18655 ±1032 3193 ±265 rNPC1L1(C17)751 ±74 202 ±10 5379 ±481 1510 ±62 rNPC1L1(C7) 462 ±25 191 ±54 3597 ±1931496 ±403 Free Cholesterol, dpm ±sem Free Cholesterol, dpm/mg protein±sem Vehicle EZ (10 μM) Vehicle EZ (10 μM) 4 Hour Pulse CHO Control61612 ±1227 56792 ±568 533876 ±17770 519607 ±16203 SR-B1 214678 ±4241194519 ±474 1762873 ±46607 1521341 ±4185 mNPC1L1(C7) 79628 ±793 77516±1910 544661 ±1269 523803 ±10386 rNPC1L1(C21) 71352 ±1343 69106 ±711498016 ±8171 485460 ±4410 rNPC1L1(C17) 78956 ±3782 71646 ±446 566456±29204 536651 ±7146 rNPC1L1(C7) 75348 ±2093 70628 ±212 586127 ±13932556855 ±7481

[0219] TABLE 6 Uptake of labeled cholesterol in the presence ofincreasing amounts of unlabeled cholesterol. Total Cholesterol, dpm ±semCHO Control SR-B1 mNPC1L1(C7) rNPC1L1(C21) Cold Cholesterol 24 Min Pulse 3 μg/mL  12271 ± 430  49603 ± 2428   1450 ± 1628  10656 ± 1233  10μg/mL  16282 ± 2438  79967 ± 8151  25465 ± 3037  13225 ± 4556  30 μg/mL 14758 ± 1607  71925 ± 3861  19001 ± 1530  13218 ± 1149 100 μg/mL  16458± 1614  58185 ± 4548  15973 ± 1665  11560 ± 1132 Cholesteryl Ester, dpm±sem CHO Control SR-B1 mNPC1L1(C7) rNPC1L1(C21) 4 Hour Pulse  3 μg/mL  2737 ± 114  39596 ± 1241   1561 ± 1   4015 ± 47  10 μg/mL   1646 ± 76 17292 ± 362   998 ± 36   1866 ± 33  30 μg/mL   970 ± 46   6642 ± 153  537 ± 82   970 ± 9 100 μg/mL   895 ± 156   4777 ± 27   405 ± 7   777 ±16 Free Cholesterol, dpm ±sem CHO Control SR-B1 mNPC1L1(C7) rNPC1L1(C21)4 Hour Pulse  3 μg/mL  89013 ± 3724  211783 ± 3268  104343 ± 2112  92244± 987  10 μg/mL  136396 ± 8566  278216 ± 10901  196173 ± 4721  125144 ±877  30 μg/mL  13174 ± 2922  224429 ± 2556  149172 ± 19689  117143 ±4976 100 μg/mL  79336 ± 4011  231470 ± 4221  114599 ± 2803  93538 ± 1588Cholesteryl Ester, dpm ±sem CHO Control SR-B1 mNPC1L1(C7) rNPC1L1(C21)24 Hour Pulse  3 μg/mL  57373 ± 2704  162296 ± 1644  22986 ± 940  59377± 953  10 μg/mL  33730 ± 1296  112815 ± 373  14836 ± 552  31797 ± 525 30 μg/mL  19193 ± 100  58668 ± 1413   8878 ± 355  18963 ± 380 100 μg/mL 16761 ± 398  31280 ± 1270   8784 ± 946  14933 ± 311 Free Cholesterol,dpm ±sem CHO Control SR-B1 mNPC1L1(C7) rNPC1L1(C21) 24 Hour Pulse  3μg/mL  248985 ± 4207  357819 ± 4519  285610 ± 5187  227244 ± 1016  10μg/mL  231208 ± 8927  269822 ± 5872  311777 ± 8227  231666 ± 6198  30μg/mL  203566 ± 6008  225273 ± 5932  279604 ± 6612  209372 ± 3386 100μg/mL  178424 ± 2379  167082 ± 2211  229832 ± 4199  182678 ± 7709 TotalCholesterol, dpm/mg protein ±sem CHO Control SR-B1 mNPC1L1(C7)rNPC1L1(C21) Cold Cholesterol 24 Min Pulse  3 μg/mL  108936 ± 5413 541562 ± 13785  140764 ± 14433  94945 ± 12916  10 μg/mL  151283 ± 23345 880224 ± 82254  250985 ± 27481  123433 ± 34092  30 μg/mL  135109 ±12106  796236 ± 18952  180436 ± 12112  111522 ± 6941 100 μg/mL  149559 ±17977  630143 ± 3718  147717 ± 8261  101328 ± 7191 Cholesteryl Ester,dpm/mg protein ±sem CHO Control SR-B1 mNPC1L1(C7) rNPC1L1(C21) 4 HourPulse  3 μg/mL  22050 ± 978  382641 ± 5955  13684 ± 217  32020 ± 641  10μg/mL  13323 ± 606  157914 ± 3400   8917 ± 467  14849 ± 127  30 μg/mL  7627 ± 323  63547 ± 1760   4885 ± 748   7741 ± 100 100 μg/mL   7135 ±1230  45088 ± 1526   3663 ± 68   6005 ± 198 Free Cholesterol, dpm/mgprotein ±sem CHO Control SR-B1 mNPC1L1(C7) rNPC1L1(C21) 4 Hour Pulse  3μg/mL  717308 ± 34130 2047695 ± 16213  914107 ± 5869  735498 ± 11209  10μg/mL 1105118 ± 76074 2540130 ± 92471 1753072 ± 86578  996824 ± 27850 30 μg/mL 1036195 ± 21142 2149215 ± 78068 1357136 ± 180264  934772 ±43202 100 μg/mL  632965 ± 29756 2182022 ± 36793 1035979 ± 30329  723225± 21694 Cholesteryl Ester, dpm/mg protein ±sem CHO Control SR-B1mNPC1L1(C7) rNPC1L1(C21) 24 Hour Pulse  3 μg/mL  357629 ± 14639 1248900± 18565  160328 ± 6565 4031315 ± 5557  10 μg/mL  215004 ± 5942  830231 ±12764  98594 ± 4205  200451 ± 5239  30 μg/mL  122071 ± 1271  446581 ±3472  59091 ± 2697  119728 ± 2131 100 μg/mL  103235 ± 1739  272796 ±13392  60670 ± 4597  96215 ± 1023 Free Cholesterol, dpm/mg protein ±semCHO Control SR-B1 mNPC1L1(C7) rNPC1L1(C21) 24 Hour Pulse  3 μg/mL1552637 ± 18954 2752957 ± 24984 1993256 ± 56968 1536023 ± 10304  10μg/mL 1477414 ± 85954 1984473 ± 18420 2069980 ± 25517 1461157 ± 58517 30 μg/mL 1294878 ± 41819 1716066 ± 52581 1859476 ± 29507 1321730 ± 5452100 μg/mL 1099648 ± 25160 1455799 ± 9885 1599244 ± 76938 1177546 ± 51191

Example 20 Labeled Cholesterol Uptake Assay

[0220] In this example, the ability of CHO cells transiently transfectedwith rat NPC1L1 or mouse SR-B1 to take up labeled cholesterol wasevaluated. Also evaluated was the ability of rat NPC1L1 to potentiatethe ability of CHO cells transfected with mouse SR-B1 to take up labeledcholesterol. In these assays, cholesterol uptake, at a singleconcentration, was evaluated in pulse-chase experiments. The datagenerated in these experiments are set forth, below, in Table 7.

[0221] Cells:

[0222] A. CHO background cells (mock transfection).

[0223] B. CHO cells transiently transfected with mouse SR-B1.

[0224] C. CHO transiently transfected with rat NPC1L1 cDNAs (n=8clones).

[0225] Transiently transfected cells were seeded at 300,000 cells/well(mL) in 12-well plates.

[0226] Procedure:

[0227] All reagents and culture plates were maintained at 37° C. unlessotherwise noted.

[0228] Starve. The maintenance media (F12 HAMS, 1% Pen/Strep, 10% FCS)was removed from the cells and replaced with 1 mL “starve” media (F12HAMS, Pen/Strep, 5% lipoprotein deficient serum (LPDS). Cells werestarved for 1 hour.

[0229]³H-Cholesterol Pulse. The following was added directly to eachwell.

[0230] 0.5 μCi ³H-cholesterol (1.1×10⁶ dpm/well) in 50 μl of a mixedbile salt micelle.

[0231] 4.8 mM sodium taurocholate (2.581 mg/mL)

[0232] 0.6 mM sodium oleate (0.183 mg/mL)

[0233] 0.25 mM cholesterol (0.1 mg/mL)

[0234] Dispersed in “starve” media by ultrasonic vibration

[0235] Final media cholesterol concentration=5 μg/mL

[0236] Labeled cholesterol pulse time points were 24 Min and 4 hours.Triplicate wells for each treatment.

[0237] Wash. At the designated times, media was aspirated and cells werewashed once with Hobbs Buffer A (50 mM Tris, 0.9% NaCl, 0.2% BSA, pH7.4) and once with Hobbs Buffer B (50 mM Tris, 0.9% NaCl, pH 7.4 (noBSA)) at 37° C.

[0238] Processing/Analysis.

[0239] A. 24 minute time point: Cells were digested overnight with 0.2NNaOH, 2 mL/well at room temp. One, 1.5 mL aliquot was removed from eachwell, neutralized & counted for radioactivity by scintillation counting.

[0240] B. 4 hour time point: The digested cells were analyzed bythin-layer chromatography to determine the content of cholesterol esterin the cells.

[0241] The extracts were spotted onto thin layer chromatography platesand run in 2 ml hexane:isopropanol (3:2) containing mobile phase for 30minutes, followed by a second run in 1 ml hexane:isopropanol (3:2)containing mobile phase for 15 min.

[0242] C. Protein determination of cell extracts: Plates containing asample of the cell extracts were placed on orbital shaker at 120 rpm forindicated times and then extracts are pooled into 12×75 tubes. Plateswere dried and NaOH (2 ml/well) added. The protein content of thesamples were then determined. Two additional 50 μl aliquots from allwells were assayed for total protein by the Pierce micro BCA method. Thequantity of labeled cholesterol observed in the cells was normalized tothe quantity of protein in the cells. TABLE 7 Labeled cholesterol uptakein transiently transfected CHO cells. Transfection dpm dpm/mg proteinTotal Cholesterol, ±sem 24 Min Pulse CHO Control (mock)  4721 ± 436 49024 ± 4328 SR-BI(Transient)  5842 ± 82  59445 ± 1099 NPC1L1(Transient)  4092 ± 377  47026 ± 2658 SR-BI/NPC1L1 (trans)  3833 ± 158 52132 ± 3071 Cholesteryl Ester, ±sem 4 Hour Pulse CHO Control (mock) 2132 ± 40  20497 ± 640 SR-BI(Transient)  5918 ± 237  51812 ± 1417NPC1L1 (Transient)  1944 ± 93  19788 ± 642 SR-BI/NPC1L1 (trans)  4747 ±39  58603 ± 1156 Free Cholesterol, ±sem 4 Hour Pulse CHO Control (mock)45729 ± 328 439346 ± 5389 SR-BI(Transient) 50820 ± 2369 444551 ± 9785NPC1L1 (Transient) 39913 ± 1211 406615 ± 6820 SR-BI/NPC1L1 (trans) 37269± 1225 459509 ± 6195

Example 21 Expression of Rat, Mouse and Human NPC1L1

[0243] In this example, NPC1L1 was introduced into cells and expressed.Species specific NPC1L1 expression constructs were cloned into theplasmid pcDNA3 using clone specific PCR primers to generate the ORFflanked by appropriate restriction sites compatible with the polylinkerof the vector. For all three species of NPC1L1, small intestine totaltissue RNA was used as a template for reverse transcriptase-polymerasechain reaction (RT-PCR) using oligo dT as the template primer. The ratNPC1L1 was cloned as an EcoRI fragment, human NPC1L1 was cloned as aXbaI/NotI fragment and mouse NPC1L1 was cloned as an EcoRI fragment.Forward and reverse strand sequencing of each clone was performed toconfirm sequence integrity. Standard transient transfection procedureswere used with CHO cells. In a 6-well plate CHO cells were plated 1 daybefore transfection at a plating density of 2×10⁵ cells/well. Thefollowing day, cells were incubated with 2 μg plasmid DNA and 6 μLLipofectamine for 5 hours followed a fresh media change. Forty-eighthours later, cells were analyzed for NPC1L1 expression using anti-NPC1L1antisera by either FACS or western blot. To establish stable long termcell lines expressing NPC1L1, transfected CHO cells were selected in thepresence of geneticin (G418, 0.8 mg/ml) as recommended by themanufacturer (Life Technologies). Following one month of selection inculture, the cell population was stained with anti-NPC1L1 antisera andsorted by FACS. Individual positive staining cells were cloned afterisolation by limiting dilution and then maintained in selective mediacontaining geneticin (0.5 mg/ml).

[0244] Other cell types less susceptible to transfection procedures havebeen generated using adenoviral vector systems. This system used toexpress NPC1L1 is dervied from Ad 5, a type C adenovirus. Thisrecombinant replication-defective adenoviral vector is made defectivethrough modifications of the E1, E2 and E4 regions. The vector also hasadditional modifications to the E3 region generally affecting the E3bregion genes RIDa and RIDb. NPC1L1 expression was driven using the CMVpromoter as an expression cassette substituted in the E3 region of theadenovirus. Rat and mouse NPC1L1 were amplified using clone specificprimers flanked by restriction sites compatible with the adenovirusvector Adenovirus infective particles were produced from 293-D22 cellsin titers of 5×10¹⁰ P/mL. Viral lysates were used to infect cellsresistant to standard transfection methodologies. In Caco2 cells, whichare highly resistant to heterologous protein expression, adenovirusmediated expression of NPC1L1 has been shown by western blot analysis topersist at least 21 days post-infection.

Example 22 NPC1L1 Knock-Out Transgenic Mouse.

[0245] NPC1L1 knockout mice were constructed via targeted mutagenesis.This methodology utilized a targeting construct designed to delete aspecific region of the mouse NPC1L1 gene. During the targeting processthe E. coli lacZ reporter gene was inserted under the control of theendogenous NPC1L1 promoter. The region in NPC1L1 (SEQ ID NO: 45) beingdeleted is from nucleotide 790 to nucleotide 998. The targeting vectorcontains the LacZ-Neo cassette flanked by 1.9 kb 5′ arm ending withnucleotide 789 and a 3.2 kb 3′ arm starting with nucleotide 999. GenomicDNA from the recombinant embryonic stem cell line was assayed forhomologous recombination using PCR. Amplified DNA fragments werevisualized by agarose gel electrophoresis. The test PCRs employed a genespecific primer, which lies outside of and adjacent to the targetingvector arm, paired with one of three primers specific to the LacZ-Neocassette sequence. For 5′ PCR reconfirmation, the NPC1L1 specificoligonucleotide ATGTTAGGTGAGTCTGAACCTACCC (SEQ ID NO: 46) and for 3′PCRreconfirmation the NPC1L1 specific oligonucleotide GGATTGCATTTCCTTCAAGAAAGCC (SEQ ID NO: 47) were used. Genotyping of the F2 mice wasperformed by multiplex PCR using the NPC1L1 specific forward primerTATGGCTCTGCCC TCTGCAATGCTC (SEQ ID NO: 48) the LacZ-Neo cassettespecific forward primer TCAGCAGCCTCTGTTCCACATACACTTC (SEQ ID NO: 49) incombination with the NPC1L1 gene specific reverse primerGTTCCACAGGGTCTGTGGTGAGTTC (SEQ ID NO: 50) allowed for determination ofboth the targeted and endogenous alleles. Analysis of the PCR productsby agarose gel electrophoresis distinguished the wild-type, heterozygoteand homozygote null mouse from each other.

[0246] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

[0247] Patents, patent applications, publications, product descriptions,Genbank Accession Numbers and protocols are cited throughout thisapplication, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

1 50 1 3996 DNA Rattus sp. CDS (1)..(3996) 1 atg gca gct gcc tgg ctg ggatgg ctg ctc tgg gcc ctg ctc ctg agc 48 Met Ala Ala Ala Trp Leu Gly TrpLeu Leu Trp Ala Leu Leu Leu Ser 1 5 10 15 gcg gcc cag ggt gag cta tacaca ccc aaa cac gaa gct ggg gtc tgc 96 Ala Ala Gln Gly Glu Leu Tyr ThrPro Lys His Glu Ala Gly Val Cys 20 25 30 acc ttt tac gaa gag tgc ggg aaaaac cca gag ctc tct gga ggc ctc 144 Thr Phe Tyr Glu Glu Cys Gly Lys AsnPro Glu Leu Ser Gly Gly Leu 35 40 45 acg tca cta tcc aat gta tcc tgc ctgtct aac acc ccg gcc cgc cac 192 Thr Ser Leu Ser Asn Val Ser Cys Leu SerAsn Thr Pro Ala Arg His 50 55 60 gtc acg ggt gaa cac ctg gct ctt ctc cagcgc atc tgt ccc cgc ctg 240 Val Thr Gly Glu His Leu Ala Leu Leu Gln ArgIle Cys Pro Arg Leu 65 70 75 80 tac aac ggc ccc aat acc act ttt gcc tgttgc tct acc aag cag ctg 288 Tyr Asn Gly Pro Asn Thr Thr Phe Ala Cys CysSer Thr Lys Gln Leu 85 90 95 ctg tcc tta gaa agc agc atg tcc atc acc aaggcc ctt ctc acg cgc 336 Leu Ser Leu Glu Ser Ser Met Ser Ile Thr Lys AlaLeu Leu Thr Arg 100 105 110 tgc ccg gcc tgc tct gac aat ttt gtg agc ttacac tgc cac aac act 384 Cys Pro Ala Cys Ser Asp Asn Phe Val Ser Leu HisCys His Asn Thr 115 120 125 tgc agc cct gac cag agc ctc ttc atc aac gtcacc cgg gtg gtt gag 432 Cys Ser Pro Asp Gln Ser Leu Phe Ile Asn Val ThrArg Val Val Glu 130 135 140 cgg ggc gct gga gag cct cct gcc gtg gtg gcctat gag gcc ttt tat 480 Arg Gly Ala Gly Glu Pro Pro Ala Val Val Ala TyrGlu Ala Phe Tyr 145 150 155 160 cag cgc agc ttt gct gag aag gcc tat gagtcc tgc agc cag gtg cgc 528 Gln Arg Ser Phe Ala Glu Lys Ala Tyr Glu SerCys Ser Gln Val Arg 165 170 175 atc cct gcg gcc gct tcc ttg gcc gtg ggcagc atg tgt gga gtg tat 576 Ile Pro Ala Ala Ala Ser Leu Ala Val Gly SerMet Cys Gly Val Tyr 180 185 190 ggc tcc gcc ctc tgc aat gct cag cgc tggctc aac ttc caa gga gac 624 Gly Ser Ala Leu Cys Asn Ala Gln Arg Trp LeuAsn Phe Gln Gly Asp 195 200 205 aca ggg aat ggc ctg gct ccg ctg gat atcacc ttc cac ctc ttg gag 672 Thr Gly Asn Gly Leu Ala Pro Leu Asp Ile ThrPhe His Leu Leu Glu 210 215 220 cct ggc cag gcc cta ccg gat ggg atc cagcca ctg aat ggg aag atc 720 Pro Gly Gln Ala Leu Pro Asp Gly Ile Gln ProLeu Asn Gly Lys Ile 225 230 235 240 gca ccc tgc aac gag tct cag ggt gatgac tca gca gtc tgc tcc tgc 768 Ala Pro Cys Asn Glu Ser Gln Gly Asp AspSer Ala Val Cys Ser Cys 245 250 255 cag gac tgt gcg gcg tcc tgc cct gtcatc cct ccg ccc gag gcc ttg 816 Gln Asp Cys Ala Ala Ser Cys Pro Val IlePro Pro Pro Glu Ala Leu 260 265 270 cgc cct tcc ttc tac atg ggt cgc atgcca ggc tgg ctg gcc ctc atc 864 Arg Pro Ser Phe Tyr Met Gly Arg Met ProGly Trp Leu Ala Leu Ile 275 280 285 atc atc ttc act gct gtc ttt gtg ttgctc tct gca gtc ctt gtg cgt 912 Ile Ile Phe Thr Ala Val Phe Val Leu LeuSer Ala Val Leu Val Arg 290 295 300 ctc cga gtg gtt tcc aac agg aac aagaac aag gca gaa ggc ccc cag 960 Leu Arg Val Val Ser Asn Arg Asn Lys AsnLys Ala Glu Gly Pro Gln 305 310 315 320 gaa gcc ccc aaa ctc cct cat aagcac aaa ctc tca ccc cat acc atc 1008 Glu Ala Pro Lys Leu Pro His Lys HisLys Leu Ser Pro His Thr Ile 325 330 335 ctg ggc cgg ttc ttc cag aac tggggc aca agg gtg gcc tcg tgg cca 1056 Leu Gly Arg Phe Phe Gln Asn Trp GlyThr Arg Val Ala Ser Trp Pro 340 345 350 ctc acc gtc tta gca ctg tcc ttcatc gtt gtg ata gcc tta gca gca 1104 Leu Thr Val Leu Ala Leu Ser Phe IleVal Val Ile Ala Leu Ala Ala 355 360 365 ggc ctg acc ttt att gaa ctc accaca gac cct gtg gaa ctg tgg tcg 1152 Gly Leu Thr Phe Ile Glu Leu Thr ThrAsp Pro Val Glu Leu Trp Ser 370 375 380 gcc ccc aag agc cag gcc cgg aaagag aag tct ttc cat gat gag cat 1200 Ala Pro Lys Ser Gln Ala Arg Lys GluLys Ser Phe His Asp Glu His 385 390 395 400 ttc ggc ccc ttc ttt cga accaac cag att ttc gtg aca gct cgg aac 1248 Phe Gly Pro Phe Phe Arg Thr AsnGln Ile Phe Val Thr Ala Arg Asn 405 410 415 agg tcc agc tac aag tac gactcc cta ctg cta ggg tcc aag aac ttc 1296 Arg Ser Ser Tyr Lys Tyr Asp SerLeu Leu Leu Gly Ser Lys Asn Phe 420 425 430 agt ggg atc ctg tcc ctg gacttc ctg ctg gag ctg ctg gag ctt cag 1344 Ser Gly Ile Leu Ser Leu Asp PheLeu Leu Glu Leu Leu Glu Leu Gln 435 440 445 gag agg ctt cga cac ctg caagtg tgg tcc cct gag gca gag cgc aac 1392 Glu Arg Leu Arg His Leu Gln ValTrp Ser Pro Glu Ala Glu Arg Asn 450 455 460 atc tcc ctc cag gac atc tgctat gcc ccc ctc aac cca tat aac acc 1440 Ile Ser Leu Gln Asp Ile Cys TyrAla Pro Leu Asn Pro Tyr Asn Thr 465 470 475 480 agc ctc tcc gac tgc tgtgtc aac agc ctc ctt cag tac ttc cag aac 1488 Ser Leu Ser Asp Cys Cys ValAsn Ser Leu Leu Gln Tyr Phe Gln Asn 485 490 495 aac cgc acc ctc ctg atgctc acg gcc aac cag act ctg aat ggc cag 1536 Asn Arg Thr Leu Leu Met LeuThr Ala Asn Gln Thr Leu Asn Gly Gln 500 505 510 acc tcc ctg gtg gac tggaag gac cat ttc ctc tac tgt gca aat gcc 1584 Thr Ser Leu Val Asp Trp LysAsp His Phe Leu Tyr Cys Ala Asn Ala 515 520 525 cct ctc acg ttc aaa gatggc acg tct ctg gcc ctg agc tgc atg gct 1632 Pro Leu Thr Phe Lys Asp GlyThr Ser Leu Ala Leu Ser Cys Met Ala 530 535 540 gac tac ggg gct cct gtcttc ccc ttc ctt gct gtt ggg gga tac caa 1680 Asp Tyr Gly Ala Pro Val PhePro Phe Leu Ala Val Gly Gly Tyr Gln 545 550 555 560 ggc acg gac tat tccgag gca gaa gcg ctg atc ata acc ttc tct ctc 1728 Gly Thr Asp Tyr Ser GluAla Glu Ala Leu Ile Ile Thr Phe Ser Leu 565 570 575 aat aac tac ccc gctgat gat ccc cgc atg gcc cag gcc aag ctc tgg 1776 Asn Asn Tyr Pro Ala AspAsp Pro Arg Met Ala Gln Ala Lys Leu Trp 580 585 590 gag gag gct ttc ttgaag gaa atg gaa tcc ttc cag agg aac aca agt 1824 Glu Glu Ala Phe Leu LysGlu Met Glu Ser Phe Gln Arg Asn Thr Ser 595 600 605 gac aag ttc cag gttgcg ttc tca gct gag cgc tct ctg gag gat gag 1872 Asp Lys Phe Gln Val AlaPhe Ser Ala Glu Arg Ser Leu Glu Asp Glu 610 615 620 atc aac cgc acc accatc cag gac ctg cct gtc ttt gcc gtc agc tac 1920 Ile Asn Arg Thr Thr IleGln Asp Leu Pro Val Phe Ala Val Ser Tyr 625 630 635 640 att atc gtc ttcctg tac atc tcc ctg gcc ctg ggc agc tac tcc aga 1968 Ile Ile Val Phe LeuTyr Ile Ser Leu Ala Leu Gly Ser Tyr Ser Arg 645 650 655 tgc agc cga gtagcg gtg gag tcc aag gct act ctg ggc cta ggt ggg 2016 Cys Ser Arg Val AlaVal Glu Ser Lys Ala Thr Leu Gly Leu Gly Gly 660 665 670 gtg att gtt gtgctg gga gca gtt ctg gct gcc atg ggc ttc tac tcc 2064 Val Ile Val Val LeuGly Ala Val Leu Ala Ala Met Gly Phe Tyr Ser 675 680 685 tac ctg ggt gtcccc tct tct ctg gtt atc atc caa gtg gta cct ttc 2112 Tyr Leu Gly Val ProSer Ser Leu Val Ile Ile Gln Val Val Pro Phe 690 695 700 ctg gtg cta gctgtg gga gct gac aac atc ttc atc ttt gtt ctt gag 2160 Leu Val Leu Ala ValGly Ala Asp Asn Ile Phe Ile Phe Val Leu Glu 705 710 715 720 tac cag aggcta cct agg atg cct ggg gaa cag cga gag gct cac att 2208 Tyr Gln Arg LeuPro Arg Met Pro Gly Glu Gln Arg Glu Ala His Ile 725 730 735 ggc cgc accctg ggc agt gtg gcc ccc agc atg ctg ctg tgc agc ctc 2256 Gly Arg Thr LeuGly Ser Val Ala Pro Ser Met Leu Leu Cys Ser Leu 740 745 750 tct gag gccatc tgc ttc ttt cta ggg gcc ctg acc ccc atg cca gct 2304 Ser Glu Ala IleCys Phe Phe Leu Gly Ala Leu Thr Pro Met Pro Ala 755 760 765 gtg agg accttc gcc ttg acc tct ggc tta gca att atc ctc gac ttc 2352 Val Arg Thr PheAla Leu Thr Ser Gly Leu Ala Ile Ile Leu Asp Phe 770 775 780 ctg ctc cagatg act gcc ttt gtg gcc ctg ctc tcc ctg gat agc aag 2400 Leu Leu Gln MetThr Ala Phe Val Ala Leu Leu Ser Leu Asp Ser Lys 785 790 795 800 agg caggag gcc tct cgc ccg gat gtc tta tgc tgc ttt tca acc cgg 2448 Arg Gln GluAla Ser Arg Pro Asp Val Leu Cys Cys Phe Ser Thr Arg 805 810 815 aag ctgccc cca cct aaa gaa aaa gaa ggc ctc tta ctc cgc ttc ttc 2496 Lys Leu ProPro Pro Lys Glu Lys Glu Gly Leu Leu Leu Arg Phe Phe 820 825 830 cgc aagata tac gct cct ttc ctg ctg cac aga ttc atc cgc cct gtt 2544 Arg Lys IleTyr Ala Pro Phe Leu Leu His Arg Phe Ile Arg Pro Val 835 840 845 gtg atgctg ctg ttt ctg acc ctg ttt gga gca aat ctc tac tta atg 2592 Val Met LeuLeu Phe Leu Thr Leu Phe Gly Ala Asn Leu Tyr Leu Met 850 855 860 tgc aacatc aac gtg ggg cta gac cag gag ctg gct ctg ccc aag gac 2640 Cys Asn IleAsn Val Gly Leu Asp Gln Glu Leu Ala Leu Pro Lys Asp 865 870 875 880 tcgtac ttg ata gac tac ttc ctc ttt ctg aac cga tac ctt gaa gtg 2688 Ser TyrLeu Ile Asp Tyr Phe Leu Phe Leu Asn Arg Tyr Leu Glu Val 885 890 895 gggcct cca gtg tac ttt gtc acc acc tcg ggc ttc aac ttc tcc agc 2736 Gly ProPro Val Tyr Phe Val Thr Thr Ser Gly Phe Asn Phe Ser Ser 900 905 910 gaggca ggc atg aac gcc act tgc tct agc gca ggc tgt aag agc ttc 2784 Glu AlaGly Met Asn Ala Thr Cys Ser Ser Ala Gly Cys Lys Ser Phe 915 920 925 tcccta acc cag aaa atc cag tat gcc agt gaa ttc cct gac cag tct 2832 Ser LeuThr Gln Lys Ile Gln Tyr Ala Ser Glu Phe Pro Asp Gln Ser 930 935 940 tacgtg gct att gct gca tcc tcc tgg gta gat gac ttc atc gac tgg 2880 Tyr ValAla Ile Ala Ala Ser Ser Trp Val Asp Asp Phe Ile Asp Trp 945 950 955 960ctg acc ccg tcc tcc tcc tgc tgt cgc ctt tat ata cgt ggc ccc cat 2928 LeuThr Pro Ser Ser Ser Cys Cys Arg Leu Tyr Ile Arg Gly Pro His 965 970 975aag gat gag ttc tgt ccc tca acg gat act tcc ttc aac tgc tta aaa 2976 LysAsp Glu Phe Cys Pro Ser Thr Asp Thr Ser Phe Asn Cys Leu Lys 980 985 990aac tgc atg aac cgc act ctg ggt cct gtg agg ccc aca gcg gaa cag 3024 AsnCys Met Asn Arg Thr Leu Gly Pro Val Arg Pro Thr Ala Glu Gln 995 10001005 ttt cat aag tac ctg ccc tgg ttc ctg aat gat ccg ccc aat atc 3069Phe His Lys Tyr Leu Pro Trp Phe Leu Asn Asp Pro Pro Asn Ile 1010 10151020 aga tgt ccc aaa ggg ggt cta gca gcg tat aga acg tct gtg aat 3114Arg Cys Pro Lys Gly Gly Leu Ala Ala Tyr Arg Thr Ser Val Asn 1025 10301035 ttg agc tca gat ggc cag gtt ata gcc tcc cag ttc atg gcc tac 3159Leu Ser Ser Asp Gly Gln Val Ile Ala Ser Gln Phe Met Ala Tyr 1040 10451050 cac aag ccc tta agg aac tca cag gac ttc aca gaa gct ctc cgg 3204His Lys Pro Leu Arg Asn Ser Gln Asp Phe Thr Glu Ala Leu Arg 1055 10601065 gcg tcc cgg ttg cta gca gcc aac atc aca gct gac cta cgg aag 3249Ala Ser Arg Leu Leu Ala Ala Asn Ile Thr Ala Asp Leu Arg Lys 1070 10751080 gtg cct ggg aca gat cca aac ttt gag gtc ttc cct tac acg atc 3294Val Pro Gly Thr Asp Pro Asn Phe Glu Val Phe Pro Tyr Thr Ile 1085 10901095 tcc aac gtg ttc tac cag caa tac ctg acg gtc ctt cct gag gga 3339Ser Asn Val Phe Tyr Gln Gln Tyr Leu Thr Val Leu Pro Glu Gly 1100 11051110 atc ttc acc ctt gct ctt tgc ttt gtg ccc acc ttt gtt gtc tgc 3384Ile Phe Thr Leu Ala Leu Cys Phe Val Pro Thr Phe Val Val Cys 1115 11201125 tac ctc cta ctg ggc ctg gac atg tgc tca ggg atc ctc aac cta 3429Tyr Leu Leu Leu Gly Leu Asp Met Cys Ser Gly Ile Leu Asn Leu 1130 11351140 ctc tcc atc att atg att ctc gtg gac acc att ggc ctc atg gct 3474Leu Ser Ile Ile Met Ile Leu Val Asp Thr Ile Gly Leu Met Ala 1145 11501155 gtg tgg ggt atc agc tat aat gcg gta tcc ctc atc aac ctt gtc 3519Val Trp Gly Ile Ser Tyr Asn Ala Val Ser Leu Ile Asn Leu Val 1160 11651170 acg gca gtg ggc atg tct gtg gag ttt gtg tcc cac atc act cgg 3564Thr Ala Val Gly Met Ser Val Glu Phe Val Ser His Ile Thr Arg 1175 11801185 tcc ttt gct gta agc acc aag cct acc cgg ctg gag agg gct aaa 3609Ser Phe Ala Val Ser Thr Lys Pro Thr Arg Leu Glu Arg Ala Lys 1190 11951200 gat gct act gtc ttc atg ggc agt gcg gtg ttt gct gga gtg gcc 3654Asp Ala Thr Val Phe Met Gly Ser Ala Val Phe Ala Gly Val Ala 1205 12101215 atg acc aac ttc cca ggc atc ctc atc ttg ggc ttt gcc caa gcc 3699Met Thr Asn Phe Pro Gly Ile Leu Ile Leu Gly Phe Ala Gln Ala 1220 12251230 cag ctt att cag atc ttc ttc ttc cgc ctc aac ctt ctg atc acc 3744Gln Leu Ile Gln Ile Phe Phe Phe Arg Leu Asn Leu Leu Ile Thr 1235 12401245 ttg ctg ggt ctg ctg cat ggc ctg gtc ttc ctg ccg gtt gtc ctc 3789Leu Leu Gly Leu Leu His Gly Leu Val Phe Leu Pro Val Val Leu 1250 12551260 agc tat ctg gga cca gat gtt aac caa gct ctg gta cag gag gag 3834Ser Tyr Leu Gly Pro Asp Val Asn Gln Ala Leu Val Gln Glu Glu 1265 12701275 aaa cta gcc agc gag gca gca gtg gcc cca gag cct tct tgc cca 3879Lys Leu Ala Ser Glu Ala Ala Val Ala Pro Glu Pro Ser Cys Pro 1280 12851290 cag tac ccc tcc cct gct gat gcg gat gcc aat gtt aac tac ggc 3924Gln Tyr Pro Ser Pro Ala Asp Ala Asp Ala Asn Val Asn Tyr Gly 1295 13001305 ttt gcc cca gaa ctt gcc cac gga gct aat gct gct aga agc tct 3969Phe Ala Pro Glu Leu Ala His Gly Ala Asn Ala Ala Arg Ser Ser 1310 13151320 ttg ccc aaa agt gac caa aag ttc taa 3996 Leu Pro Lys Ser Asp GlnLys Phe 1325 1330 2 1331 PRT Rattus sp. 2 Met Ala Ala Ala Trp Leu GlyTrp Leu Leu Trp Ala Leu Leu Leu Ser 1 5 10 15 Ala Ala Gln Gly Glu LeuTyr Thr Pro Lys His Glu Ala Gly Val Cys 20 25 30 Thr Phe Tyr Glu Glu CysGly Lys Asn Pro Glu Leu Ser Gly Gly Leu 35 40 45 Thr Ser Leu Ser Asn ValSer Cys Leu Ser Asn Thr Pro Ala Arg His 50 55 60 Val Thr Gly Glu His LeuAla Leu Leu Gln Arg Ile Cys Pro Arg Leu 65 70 75 80 Tyr Asn Gly Pro AsnThr Thr Phe Ala Cys Cys Ser Thr Lys Gln Leu 85 90 95 Leu Ser Leu Glu SerSer Met Ser Ile Thr Lys Ala Leu Leu Thr Arg 100 105 110 Cys Pro Ala CysSer Asp Asn Phe Val Ser Leu His Cys His Asn Thr 115 120 125 Cys Ser ProAsp Gln Ser Leu Phe Ile Asn Val Thr Arg Val Val Glu 130 135 140 Arg GlyAla Gly Glu Pro Pro Ala Val Val Ala Tyr Glu Ala Phe Tyr 145 150 155 160Gln Arg Ser Phe Ala Glu Lys Ala Tyr Glu Ser Cys Ser Gln Val Arg 165 170175 Ile Pro Ala Ala Ala Ser Leu Ala Val Gly Ser Met Cys Gly Val Tyr 180185 190 Gly Ser Ala Leu Cys Asn Ala Gln Arg Trp Leu Asn Phe Gln Gly Asp195 200 205 Thr Gly Asn Gly Leu Ala Pro Leu Asp Ile Thr Phe His Leu LeuGlu 210 215 220 Pro Gly Gln Ala Leu Pro Asp Gly Ile Gln Pro Leu Asn GlyLys Ile 225 230 235 240 Ala Pro Cys Asn Glu Ser Gln Gly Asp Asp Ser AlaVal Cys Ser Cys 245 250 255 Gln Asp Cys Ala Ala Ser Cys Pro Val Ile ProPro Pro Glu Ala Leu 260 265 270 Arg Pro Ser Phe Tyr Met Gly Arg Met ProGly Trp Leu Ala Leu Ile 275 280 285 Ile Ile Phe Thr Ala Val Phe Val LeuLeu Ser Ala Val Leu Val Arg 290 295 300 Leu Arg Val Val Ser Asn Arg AsnLys Asn Lys Ala Glu Gly Pro Gln 305 310 315 320 Glu Ala Pro Lys Leu ProHis Lys His Lys Leu Ser Pro His Thr Ile 325 330 335 Leu Gly Arg Phe PheGln Asn Trp Gly Thr Arg Val Ala Ser Trp Pro 340 345 350 Leu Thr Val LeuAla Leu Ser Phe Ile Val Val Ile Ala Leu Ala Ala 355 360 365 Gly Leu ThrPhe Ile Glu Leu Thr Thr Asp Pro Val Glu Leu Trp Ser 370 375 380 Ala ProLys Ser Gln Ala Arg Lys Glu Lys Ser Phe His Asp Glu His 385 390 395 400Phe Gly Pro Phe Phe Arg Thr Asn Gln Ile Phe Val Thr Ala Arg Asn 405 410415 Arg Ser Ser Tyr Lys Tyr Asp Ser Leu Leu Leu Gly Ser Lys Asn Phe 420425 430 Ser Gly Ile Leu Ser Leu Asp Phe Leu Leu Glu Leu Leu Glu Leu Gln435 440 445 Glu Arg Leu Arg His Leu Gln Val Trp Ser Pro Glu Ala Glu ArgAsn 450 455 460 Ile Ser Leu Gln Asp Ile Cys Tyr Ala Pro Leu Asn Pro TyrAsn Thr 465 470 475 480 Ser Leu Ser Asp Cys Cys Val Asn Ser Leu Leu GlnTyr Phe Gln Asn 485 490 495 Asn Arg Thr Leu Leu Met Leu Thr Ala Asn GlnThr Leu Asn Gly Gln 500 505 510 Thr Ser Leu Val Asp Trp Lys Asp His PheLeu Tyr Cys Ala Asn Ala 515 520 525 Pro Leu Thr Phe Lys Asp Gly Thr SerLeu Ala Leu Ser Cys Met Ala 530 535 540 Asp Tyr Gly Ala Pro Val Phe ProPhe Leu Ala Val Gly Gly Tyr Gln 545 550 555 560 Gly Thr Asp Tyr Ser GluAla Glu Ala Leu Ile Ile Thr Phe Ser Leu 565 570 575 Asn Asn Tyr Pro AlaAsp Asp Pro Arg Met Ala Gln Ala Lys Leu Trp 580 585 590 Glu Glu Ala PheLeu Lys Glu Met Glu Ser Phe Gln Arg Asn Thr Ser 595 600 605 Asp Lys PheGln Val Ala Phe Ser Ala Glu Arg Ser Leu Glu Asp Glu 610 615 620 Ile AsnArg Thr Thr Ile Gln Asp Leu Pro Val Phe Ala Val Ser Tyr 625 630 635 640Ile Ile Val Phe Leu Tyr Ile Ser Leu Ala Leu Gly Ser Tyr Ser Arg 645 650655 Cys Ser Arg Val Ala Val Glu Ser Lys Ala Thr Leu Gly Leu Gly Gly 660665 670 Val Ile Val Val Leu Gly Ala Val Leu Ala Ala Met Gly Phe Tyr Ser675 680 685 Tyr Leu Gly Val Pro Ser Ser Leu Val Ile Ile Gln Val Val ProPhe 690 695 700 Leu Val Leu Ala Val Gly Ala Asp Asn Ile Phe Ile Phe ValLeu Glu 705 710 715 720 Tyr Gln Arg Leu Pro Arg Met Pro Gly Glu Gln ArgGlu Ala His Ile 725 730 735 Gly Arg Thr Leu Gly Ser Val Ala Pro Ser MetLeu Leu Cys Ser Leu 740 745 750 Ser Glu Ala Ile Cys Phe Phe Leu Gly AlaLeu Thr Pro Met Pro Ala 755 760 765 Val Arg Thr Phe Ala Leu Thr Ser GlyLeu Ala Ile Ile Leu Asp Phe 770 775 780 Leu Leu Gln Met Thr Ala Phe ValAla Leu Leu Ser Leu Asp Ser Lys 785 790 795 800 Arg Gln Glu Ala Ser ArgPro Asp Val Leu Cys Cys Phe Ser Thr Arg 805 810 815 Lys Leu Pro Pro ProLys Glu Lys Glu Gly Leu Leu Leu Arg Phe Phe 820 825 830 Arg Lys Ile TyrAla Pro Phe Leu Leu His Arg Phe Ile Arg Pro Val 835 840 845 Val Met LeuLeu Phe Leu Thr Leu Phe Gly Ala Asn Leu Tyr Leu Met 850 855 860 Cys AsnIle Asn Val Gly Leu Asp Gln Glu Leu Ala Leu Pro Lys Asp 865 870 875 880Ser Tyr Leu Ile Asp Tyr Phe Leu Phe Leu Asn Arg Tyr Leu Glu Val 885 890895 Gly Pro Pro Val Tyr Phe Val Thr Thr Ser Gly Phe Asn Phe Ser Ser 900905 910 Glu Ala Gly Met Asn Ala Thr Cys Ser Ser Ala Gly Cys Lys Ser Phe915 920 925 Ser Leu Thr Gln Lys Ile Gln Tyr Ala Ser Glu Phe Pro Asp GlnSer 930 935 940 Tyr Val Ala Ile Ala Ala Ser Ser Trp Val Asp Asp Phe IleAsp Trp 945 950 955 960 Leu Thr Pro Ser Ser Ser Cys Cys Arg Leu Tyr IleArg Gly Pro His 965 970 975 Lys Asp Glu Phe Cys Pro Ser Thr Asp Thr SerPhe Asn Cys Leu Lys 980 985 990 Asn Cys Met Asn Arg Thr Leu Gly Pro ValArg Pro Thr Ala Glu Gln 995 1000 1005 Phe His Lys Tyr Leu Pro Trp PheLeu Asn Asp Pro Pro Asn Ile 1010 1015 1020 Arg Cys Pro Lys Gly Gly LeuAla Ala Tyr Arg Thr Ser Val Asn 1025 1030 1035 Leu Ser Ser Asp Gly GlnVal Ile Ala Ser Gln Phe Met Ala Tyr 1040 1045 1050 His Lys Pro Leu ArgAsn Ser Gln Asp Phe Thr Glu Ala Leu Arg 1055 1060 1065 Ala Ser Arg LeuLeu Ala Ala Asn Ile Thr Ala Asp Leu Arg Lys 1070 1075 1080 Val Pro GlyThr Asp Pro Asn Phe Glu Val Phe Pro Tyr Thr Ile 1085 1090 1095 Ser AsnVal Phe Tyr Gln Gln Tyr Leu Thr Val Leu Pro Glu Gly 1100 1105 1110 IlePhe Thr Leu Ala Leu Cys Phe Val Pro Thr Phe Val Val Cys 1115 1120 1125Tyr Leu Leu Leu Gly Leu Asp Met Cys Ser Gly Ile Leu Asn Leu 1130 11351140 Leu Ser Ile Ile Met Ile Leu Val Asp Thr Ile Gly Leu Met Ala 11451150 1155 Val Trp Gly Ile Ser Tyr Asn Ala Val Ser Leu Ile Asn Leu Val1160 1165 1170 Thr Ala Val Gly Met Ser Val Glu Phe Val Ser His Ile ThrArg 1175 1180 1185 Ser Phe Ala Val Ser Thr Lys Pro Thr Arg Leu Glu ArgAla Lys 1190 1195 1200 Asp Ala Thr Val Phe Met Gly Ser Ala Val Phe AlaGly Val Ala 1205 1210 1215 Met Thr Asn Phe Pro Gly Ile Leu Ile Leu GlyPhe Ala Gln Ala 1220 1225 1230 Gln Leu Ile Gln Ile Phe Phe Phe Arg LeuAsn Leu Leu Ile Thr 1235 1240 1245 Leu Leu Gly Leu Leu His Gly Leu ValPhe Leu Pro Val Val Leu 1250 1255 1260 Ser Tyr Leu Gly Pro Asp Val AsnGln Ala Leu Val Gln Glu Glu 1265 1270 1275 Lys Leu Ala Ser Glu Ala AlaVal Ala Pro Glu Pro Ser Cys Pro 1280 1285 1290 Gln Tyr Pro Ser Pro AlaAsp Ala Asp Ala Asn Val Asn Tyr Gly 1295 1300 1305 Phe Ala Pro Glu LeuAla His Gly Ala Asn Ala Ala Arg Ser Ser 1310 1315 1320 Leu Pro Lys SerAsp Gln Lys Phe 1325 1330 3 3999 DNA Homo sapiens CDS (1)..(3999) 3 atggcg gag gcc ggc ctg agg ggc tgg ctg ctg tgg gcc ctg ctc ctg 48 Met AlaGlu Ala Gly Leu Arg Gly Trp Leu Leu Trp Ala Leu Leu Leu 1 5 10 15 cgcttg gcc cag agt gag cct tac aca acc atc cac cag cct ggc tac 96 Arg LeuAla Gln Ser Glu Pro Tyr Thr Thr Ile His Gln Pro Gly Tyr 20 25 30 tgc gccttc tat gac gaa tgt ggg aag aac cca gag ctg tct gga agc 144 Cys Ala PheTyr Asp Glu Cys Gly Lys Asn Pro Glu Leu Ser Gly Ser 35 40 45 ctc atg acactc tcc aac gtg tcc tgc ctg tcc aac acg ccg gcc cgc 192 Leu Met Thr LeuSer Asn Val Ser Cys Leu Ser Asn Thr Pro Ala Arg 50 55 60 aag atc aca ggtgat cac ctg atc cta tta cag aag atc tgc ccc cgc 240 Lys Ile Thr Gly AspHis Leu Ile Leu Leu Gln Lys Ile Cys Pro Arg 65 70 75 80 ctc tac acc ggcccc aac acc caa gcc tgc tgc tcc gcc aag cag ctg 288 Leu Tyr Thr Gly ProAsn Thr Gln Ala Cys Cys Ser Ala Lys Gln Leu 85 90 95 gta tca ctg gaa gcgagt ctg tcg atc acc aag gcc ctc ctc acc cgc 336 Val Ser Leu Glu Ala SerLeu Ser Ile Thr Lys Ala Leu Leu Thr Arg 100 105 110 tgc cca gcc tgc tctgac aat ttt gtg aac ctg cac tgc cac aac acg 384 Cys Pro Ala Cys Ser AspAsn Phe Val Asn Leu His Cys His Asn Thr 115 120 125 tgc agc ccc aat cagagc ctc ttc atc aat gtg acc cgc gtg gcc cag 432 Cys Ser Pro Asn Gln SerLeu Phe Ile Asn Val Thr Arg Val Ala Gln 130 135 140 cta ggg gct gga caactc cca gct gtg gtg gcc tat gag gcc ttc tac 480 Leu Gly Ala Gly Gln LeuPro Ala Val Val Ala Tyr Glu Ala Phe Tyr 145 150 155 160 cag cat agc tttgcc gag cag agc tat gac tcc tgc agc cgt gtg cgc 528 Gln His Ser Phe AlaGlu Gln Ser Tyr Asp Ser Cys Ser Arg Val Arg 165 170 175 gtc cct gca gctgcc acg ctg gct gtg ggc acc atg tgt ggc gtg tat 576 Val Pro Ala Ala AlaThr Leu Ala Val Gly Thr Met Cys Gly Val Tyr 180 185 190 ggc tct gcc ctttgc aat gcc cag cgc tgg ctc aac ttc cag gga gac 624 Gly Ser Ala Leu CysAsn Ala Gln Arg Trp Leu Asn Phe Gln Gly Asp 195 200 205 aca ggc aat ggtctg gcc cca ctg gac atc acc ttc cac ctc ttg gag 672 Thr Gly Asn Gly LeuAla Pro Leu Asp Ile Thr Phe His Leu Leu Glu 210 215 220 cct ggc cag gccgtg ggg agt ggg att cag cct ctg aat gag ggg gtt 720 Pro Gly Gln Ala ValGly Ser Gly Ile Gln Pro Leu Asn Glu Gly Val 225 230 235 240 gca cgt tgcaat gag tcc caa ggt gac gac gtg gcg acc tgc tcc tgc 768 Ala Arg Cys AsnGlu Ser Gln Gly Asp Asp Val Ala Thr Cys Ser Cys 245 250 255 caa gac tgtgct gca tcc tgt cct gcc ata gcc cgc ccc cag gcc ctc 816 Gln Asp Cys AlaAla Ser Cys Pro Ala Ile Ala Arg Pro Gln Ala Leu 260 265 270 gac tcc accttc tac ctg ggc cag atg ccg ggc agt ctg gtc ctc atc 864 Asp Ser Thr PheTyr Leu Gly Gln Met Pro Gly Ser Leu Val Leu Ile 275 280 285 atc atc ctctgc tct gtc ttc gct gtg gtc acc atc ctg ctt gtg gga 912 Ile Ile Leu CysSer Val Phe Ala Val Val Thr Ile Leu Leu Val Gly 290 295 300 ttc cgt gtggcc ccc gcc agg gac aaa agc aag atg gtg gac ccc aag 960 Phe Arg Val AlaPro Ala Arg Asp Lys Ser Lys Met Val Asp Pro Lys 305 310 315 320 aag ggcacc agc ctc tct gac aag ctc agc ttc tcc acc cac acc ctc 1008 Lys Gly ThrSer Leu Ser Asp Lys Leu Ser Phe Ser Thr His Thr Leu 325 330 335 ctt ggccag ttc ttc cag ggc tgg ggc acg tgg gtg gct tcg tgg cct 1056 Leu Gly GlnPhe Phe Gln Gly Trp Gly Thr Trp Val Ala Ser Trp Pro 340 345 350 ctg accatc ttg gtg cta tct gtc atc ccg gtg gtg gcc ttg gca gcg 1104 Leu Thr IleLeu Val Leu Ser Val Ile Pro Val Val Ala Leu Ala Ala 355 360 365 ggc ctggtc ttt aca gaa ctc act acg gac ccc gtg gag ctg tgg tcg 1152 Gly Leu ValPhe Thr Glu Leu Thr Thr Asp Pro Val Glu Leu Trp Ser 370 375 380 gcc cccaac agc caa gcc cgg agt gag aaa gct ttc cat gac cag cat 1200 Ala Pro AsnSer Gln Ala Arg Ser Glu Lys Ala Phe His Asp Gln His 385 390 395 400 ttcggc ccc ttc ttc cga acc aac cag gtg atc ctg acg gct cct aac 1248 Phe GlyPro Phe Phe Arg Thr Asn Gln Val Ile Leu Thr Ala Pro Asn 405 410 415 cggtcc agc tac agg tat gac tct ctg ctg ctg ggg ccc aag aac ttc 1296 Arg SerSer Tyr Arg Tyr Asp Ser Leu Leu Leu Gly Pro Lys Asn Phe 420 425 430 agcgga atc ctg gac ctg gac ttg ctg ctg gag ctg cta gag ctg cag 1344 Ser GlyIle Leu Asp Leu Asp Leu Leu Leu Glu Leu Leu Glu Leu Gln 435 440 445 gagagg ctg cgg cac ctc cag gta tgg tcg ccc gaa gca cag cgc aac 1392 Glu ArgLeu Arg His Leu Gln Val Trp Ser Pro Glu Ala Gln Arg Asn 450 455 460 atctcc ctg cag gac atc tgc tac gcc ccc ctc aat ccg gac aat acc 1440 Ile SerLeu Gln Asp Ile Cys Tyr Ala Pro Leu Asn Pro Asp Asn Thr 465 470 475 480agt ctc tac gac tgc tgc atc aac agc ctc ctg cag tat ttc cag aac 1488 SerLeu Tyr Asp Cys Cys Ile Asn Ser Leu Leu Gln Tyr Phe Gln Asn 485 490 495aac cgc acg ctc ctg ctg ctc aca gcc aac cag aca ctg atg ggg cag 1536 AsnArg Thr Leu Leu Leu Leu Thr Ala Asn Gln Thr Leu Met Gly Gln 500 505 510acc tcc caa gtc gac tgg aag gac cat ttt ctg tac tgt gcc aat gcc 1584 ThrSer Gln Val Asp Trp Lys Asp His Phe Leu Tyr Cys Ala Asn Ala 515 520 525ccg ctc acc ttc aag gat ggc aca gcc ctg gcc ctg agc tgc atg gct 1632 ProLeu Thr Phe Lys Asp Gly Thr Ala Leu Ala Leu Ser Cys Met Ala 530 535 540gac tac ggg gcc cct gtc ttc ccc ttc ctt gcc att ggg ggg tac aaa 1680 AspTyr Gly Ala Pro Val Phe Pro Phe Leu Ala Ile Gly Gly Tyr Lys 545 550 555560 gga aag gac tat tct gag gca gag gcc ctg atc atg acg ttc tcc ctc 1728Gly Lys Asp Tyr Ser Glu Ala Glu Ala Leu Ile Met Thr Phe Ser Leu 565 570575 aac aat tac cct gcc ggg gac ccc cgt ctg gcc cag gcc aag ctg tgg 1776Asn Asn Tyr Pro Ala Gly Asp Pro Arg Leu Ala Gln Ala Lys Leu Trp 580 585590 gag gag gcc ttc tta gag gaa atg cga gcc ttc cag cgt cgg atg gct 1824Glu Glu Ala Phe Leu Glu Glu Met Arg Ala Phe Gln Arg Arg Met Ala 595 600605 ggc atg ttc cag gtc acg ttc acg gct gag cgc tct ctg gaa gac gag 1872Gly Met Phe Gln Val Thr Phe Thr Ala Glu Arg Ser Leu Glu Asp Glu 610 615620 atc aat cgc acc aca gct gaa gac ctg ccc atc ttt gcc acc agc tac 1920Ile Asn Arg Thr Thr Ala Glu Asp Leu Pro Ile Phe Ala Thr Ser Tyr 625 630635 640 att gtc ata ttc ctg tac atc tct ctg gcc ctg ggc agc tat tcc agc1968 Ile Val Ile Phe Leu Tyr Ile Ser Leu Ala Leu Gly Ser Tyr Ser Ser 645650 655 tgg agc cga gtg atg gtg gac tcc aag gcc acg ctg ggc ctc ggc ggg2016 Trp Ser Arg Val Met Val Asp Ser Lys Ala Thr Leu Gly Leu Gly Gly 660665 670 gtg gcc gtg gtc ctg gga gca gtc atg gct gcc atg ggc ttc ttc tcc2064 Val Ala Val Val Leu Gly Ala Val Met Ala Ala Met Gly Phe Phe Ser 675680 685 tac ttg ggt atc cgc tcc tcc ctg gtc atc ctg caa gtg gtt cct ttc2112 Tyr Leu Gly Ile Arg Ser Ser Leu Val Ile Leu Gln Val Val Pro Phe 690695 700 ctg gtg ctg tcc gtg ggg gct gat aac atc ttc atc ttt gtt ctc gag2160 Leu Val Leu Ser Val Gly Ala Asp Asn Ile Phe Ile Phe Val Leu Glu 705710 715 720 tac cag agg ctg ccc cgg agg cct ggg gag cca cga gag gtc cacatt 2208 Tyr Gln Arg Leu Pro Arg Arg Pro Gly Glu Pro Arg Glu Val His Ile725 730 735 ggg cga gcc cta ggc agg gtg gct ccc agc atg ctg ttg tgc agcctc 2256 Gly Arg Ala Leu Gly Arg Val Ala Pro Ser Met Leu Leu Cys Ser Leu740 745 750 tct gag gcc atc tgc ttc ttc cta ggg gcc ctg acc ccc atg ccagct 2304 Ser Glu Ala Ile Cys Phe Phe Leu Gly Ala Leu Thr Pro Met Pro Ala755 760 765 gtg cgg acc ttt gcc ctg acc tct ggc ctt gca gtg atc ctt gacttc 2352 Val Arg Thr Phe Ala Leu Thr Ser Gly Leu Ala Val Ile Leu Asp Phe770 775 780 ctc ctg cag atg tca gcc ttt gtg gcc ctg ctc tcc ctg gac agcaag 2400 Leu Leu Gln Met Ser Ala Phe Val Ala Leu Leu Ser Leu Asp Ser Lys785 790 795 800 agg cag gag gcc tcc cgg ttg gac gtc tgc tgc tgt gtc aagccc cag 2448 Arg Gln Glu Ala Ser Arg Leu Asp Val Cys Cys Cys Val Lys ProGln 805 810 815 gag ctg ccc ccg cct ggc cag gga gag ggg ctc ctg ctt ggcttc ttc 2496 Glu Leu Pro Pro Pro Gly Gln Gly Glu Gly Leu Leu Leu Gly PhePhe 820 825 830 caa aag gct tat gcc ccc ttc ctg ctg cac tgg atc act cgaggt gtt 2544 Gln Lys Ala Tyr Ala Pro Phe Leu Leu His Trp Ile Thr Arg GlyVal 835 840 845 gtg ctg ctg ctg ttt ctc gcc ctg ttc gga gtg agc ctc tactcc atg 2592 Val Leu Leu Leu Phe Leu Ala Leu Phe Gly Val Ser Leu Tyr SerMet 850 855 860 tgc cac atc agc gtg gga ctg gac cag gag ctg gcc ctg cccaag gac 2640 Cys His Ile Ser Val Gly Leu Asp Gln Glu Leu Ala Leu Pro LysAsp 865 870 875 880 tcg tac ctg ctt gac tat ttc ctc ttt ctg aac cgc tacttc gag gtg 2688 Ser Tyr Leu Leu Asp Tyr Phe Leu Phe Leu Asn Arg Tyr PheGlu Val 885 890 895 ggg gcc ccg gtg tac ttt gtt acc acc ttg ggc tac aacttc tcc agc 2736 Gly Ala Pro Val Tyr Phe Val Thr Thr Leu Gly Tyr Asn PheSer Ser 900 905 910 gag gct ggg atg aat gcc atc tgc tcc agt gca ggc tgcaac aac ttc 2784 Glu Ala Gly Met Asn Ala Ile Cys Ser Ser Ala Gly Cys AsnAsn Phe 915 920 925 tcc ttc acc cag aag atc cag tat gcc aca gag ttc cctgag cag tct 2832 Ser Phe Thr Gln Lys Ile Gln Tyr Ala Thr Glu Phe Pro GluGln Ser 930 935 940 tac ctg gcc atc cct gcc tcc tcc tgg gtg gat gac ttcatt gac tgg 2880 Tyr Leu Ala Ile Pro Ala Ser Ser Trp Val Asp Asp Phe IleAsp Trp 945 950 955 960 ctg acc ccg tcc tcc tgc tgc cgc ctt tat ata tctggc ccc aat aag 2928 Leu Thr Pro Ser Ser Cys Cys Arg Leu Tyr Ile Ser GlyPro Asn Lys 965 970 975 gac aag ttc tgc ccc tcg acc gtc aac tct ctg aactgc cta aag aac 2976 Asp Lys Phe Cys Pro Ser Thr Val Asn Ser Leu Asn CysLeu Lys Asn 980 985 990 tgc atg agc atc acg atg ggc tct gtg agg ccc tcggtg gag cag ttc 3024 Cys Met Ser Ile Thr Met Gly Ser Val Arg Pro Ser ValGlu Gln Phe 995 1000 1005 cat aag tat ctt ccc tgg ttc ctg aac gac cggccc aac atc aaa 3069 His Lys Tyr Leu Pro Trp Phe Leu Asn Asp Arg Pro AsnIle Lys 1010 1015 1020 tgt ccc aaa ggc ggc ctg gca gca tac agc acc tctgtg aac ttg 3114 Cys Pro Lys Gly Gly Leu Ala Ala Tyr Ser Thr Ser Val AsnLeu 1025 1030 1035 act tca gat ggc cag gtt tta gcc tcc agg ttc atg gcctat cac 3159 Thr Ser Asp Gly Gln Val Leu Ala Ser Arg Phe Met Ala Tyr His1040 1045 1050 aag ccc ctg aaa aac tca cag gat tac aca gaa gct ctg cgggca 3204 Lys Pro Leu Lys Asn Ser Gln Asp Tyr Thr Glu Ala Leu Arg Ala1055 1060 1065 gct cga gag ctg gca gcc aac atc act gct gac ctg cgg aaagtg 3249 Ala Arg Glu Leu Ala Ala Asn Ile Thr Ala Asp Leu Arg Lys Val1070 1075 1080 cct gga aca gac ccg gct ttt gag gtc ttc ccc tac acg atcacc 3294 Pro Gly Thr Asp Pro Ala Phe Glu Val Phe Pro Tyr Thr Ile Thr1085 1090 1095 aat gtg ttt tat gag cag tac ctg acc atc ctc cct gag gggctc 3339 Asn Val Phe Tyr Glu Gln Tyr Leu Thr Ile Leu Pro Glu Gly Leu1100 1105 1110 ttc atg ctc agc ctc tgc ctt gtg ccc acc ttc gct gtc tcctgc 3384 Phe Met Leu Ser Leu Cys Leu Val Pro Thr Phe Ala Val Ser Cys1115 1120 1125 ctc ctg ctg ggc ctg gac ctg cgc tcc ggc ctc ctc aac ctgctc 3429 Leu Leu Leu Gly Leu Asp Leu Arg Ser Gly Leu Leu Asn Leu Leu1130 1135 1140 tcc att gtc atg atc ctc gtg gac act gtc ggc ttc atg gccctg 3474 Ser Ile Val Met Ile Leu Val Asp Thr Val Gly Phe Met Ala Leu1145 1150 1155 tgg gac atc agt tac aat gct gtg tcc ctc atc aac ctg gtctcg 3519 Trp Asp Ile Ser Tyr Asn Ala Val Ser Leu Ile Asn Leu Val Ser1160 1165 1170 gcg gtg ggc atg tct gtg gag ttt gtg tcc cac att acc cgctcc 3564 Ala Val Gly Met Ser Val Glu Phe Val Ser His Ile Thr Arg Ser1175 1180 1185 ttt gcc atc agc acc aag ccc acc tgg ctg gag agg gcc aaagag 3609 Phe Ala Ile Ser Thr Lys Pro Thr Trp Leu Glu Arg Ala Lys Glu1190 1195 1200 gcc acc atc tct atg gga agt gcg gtg ttt gca ggt gtg gccatg 3654 Ala Thr Ile Ser Met Gly Ser Ala Val Phe Ala Gly Val Ala Met1205 1210 1215 acc aac ctg cct ggc atc ctt gtc ctg ggc ctc gcc aag gcccag 3699 Thr Asn Leu Pro Gly Ile Leu Val Leu Gly Leu Ala Lys Ala Gln1220 1225 1230 ctc att cag atc ttc ttc ttc cgc ctc aac ctc ctg atc actctg 3744 Leu Ile Gln Ile Phe Phe Phe Arg Leu Asn Leu Leu Ile Thr Leu1235 1240 1245 ctg ggc ctg ctg cat ggc ttg gtc ttc ctg ccc gtc atc ctcagc 3789 Leu Gly Leu Leu His Gly Leu Val Phe Leu Pro Val Ile Leu Ser1250 1255 1260 tac gtg ggg cct gac gtt aac ccg gct ctg gca ctg gag cagaag 3834 Tyr Val Gly Pro Asp Val Asn Pro Ala Leu Ala Leu Glu Gln Lys1265 1270 1275 cgg gct gag gag gcg gtg gca gca gtc atg gtg gcc tct tgccca 3879 Arg Ala Glu Glu Ala Val Ala Ala Val Met Val Ala Ser Cys Pro1280 1285 1290 aat cac ccc tcc cga gtc tcc aca gct gac aac atc tat gtcaac 3924 Asn His Pro Ser Arg Val Ser Thr Ala Asp Asn Ile Tyr Val Asn1295 1300 1305 cac agc ttt gaa ggt tct atc aaa ggt gct ggt gcc atc agcaac 3969 His Ser Phe Glu Gly Ser Ile Lys Gly Ala Gly Ala Ile Ser Asn1310 1315 1320 ttc ttg ccc aac aat ggg cgg cag ttc tga 3999 Phe Leu ProAsn Asn Gly Arg Gln Phe 1325 1330 4 1332 PRT Homo sapiens 4 Met Ala GluAla Gly Leu Arg Gly Trp Leu Leu Trp Ala Leu Leu Leu 1 5 10 15 Arg LeuAla Gln Ser Glu Pro Tyr Thr Thr Ile His Gln Pro Gly Tyr 20 25 30 Cys AlaPhe Tyr Asp Glu Cys Gly Lys Asn Pro Glu Leu Ser Gly Ser 35 40 45 Leu MetThr Leu Ser Asn Val Ser Cys Leu Ser Asn Thr Pro Ala Arg 50 55 60 Lys IleThr Gly Asp His Leu Ile Leu Leu Gln Lys Ile Cys Pro Arg 65 70 75 80 LeuTyr Thr Gly Pro Asn Thr Gln Ala Cys Cys Ser Ala Lys Gln Leu 85 90 95 ValSer Leu Glu Ala Ser Leu Ser Ile Thr Lys Ala Leu Leu Thr Arg 100 105 110Cys Pro Ala Cys Ser Asp Asn Phe Val Asn Leu His Cys His Asn Thr 115 120125 Cys Ser Pro Asn Gln Ser Leu Phe Ile Asn Val Thr Arg Val Ala Gln 130135 140 Leu Gly Ala Gly Gln Leu Pro Ala Val Val Ala Tyr Glu Ala Phe Tyr145 150 155 160 Gln His Ser Phe Ala Glu Gln Ser Tyr Asp Ser Cys Ser ArgVal Arg 165 170 175 Val Pro Ala Ala Ala Thr Leu Ala Val Gly Thr Met CysGly Val Tyr 180 185 190 Gly Ser Ala Leu Cys Asn Ala Gln Arg Trp Leu AsnPhe Gln Gly Asp 195 200 205 Thr Gly Asn Gly Leu Ala Pro Leu Asp Ile ThrPhe His Leu Leu Glu 210 215 220 Pro Gly Gln Ala Val Gly Ser Gly Ile GlnPro Leu Asn Glu Gly Val 225 230 235 240 Ala Arg Cys Asn Glu Ser Gln GlyAsp Asp Val Ala Thr Cys Ser Cys 245 250 255 Gln Asp Cys Ala Ala Ser CysPro Ala Ile Ala Arg Pro Gln Ala Leu 260 265 270 Asp Ser Thr Phe Tyr LeuGly Gln Met Pro Gly Ser Leu Val Leu Ile 275 280 285 Ile Ile Leu Cys SerVal Phe Ala Val Val Thr Ile Leu Leu Val Gly 290 295 300 Phe Arg Val AlaPro Ala Arg Asp Lys Ser Lys Met Val Asp Pro Lys 305 310 315 320 Lys GlyThr Ser Leu Ser Asp Lys Leu Ser Phe Ser Thr His Thr Leu 325 330 335 LeuGly Gln Phe Phe Gln Gly Trp Gly Thr Trp Val Ala Ser Trp Pro 340 345 350Leu Thr Ile Leu Val Leu Ser Val Ile Pro Val Val Ala Leu Ala Ala 355 360365 Gly Leu Val Phe Thr Glu Leu Thr Thr Asp Pro Val Glu Leu Trp Ser 370375 380 Ala Pro Asn Ser Gln Ala Arg Ser Glu Lys Ala Phe His Asp Gln His385 390 395 400 Phe Gly Pro Phe Phe Arg Thr Asn Gln Val Ile Leu Thr AlaPro Asn 405 410 415 Arg Ser Ser Tyr Arg Tyr Asp Ser Leu Leu Leu Gly ProLys Asn Phe 420 425 430 Ser Gly Ile Leu Asp Leu Asp Leu Leu Leu Glu LeuLeu Glu Leu Gln 435 440 445 Glu Arg Leu Arg His Leu Gln Val Trp Ser ProGlu Ala Gln Arg Asn 450 455 460 Ile Ser Leu Gln Asp Ile Cys Tyr Ala ProLeu Asn Pro Asp Asn Thr 465 470 475 480 Ser Leu Tyr Asp Cys Cys Ile AsnSer Leu Leu Gln Tyr Phe Gln Asn 485 490 495 Asn Arg Thr Leu Leu Leu LeuThr Ala Asn Gln Thr Leu Met Gly Gln 500 505 510 Thr Ser Gln Val Asp TrpLys Asp His Phe Leu Tyr Cys Ala Asn Ala 515 520 525 Pro Leu Thr Phe LysAsp Gly Thr Ala Leu Ala Leu Ser Cys Met Ala 530 535 540 Asp Tyr Gly AlaPro Val Phe Pro Phe Leu Ala Ile Gly Gly Tyr Lys 545 550 555 560 Gly LysAsp Tyr Ser Glu Ala Glu Ala Leu Ile Met Thr Phe Ser Leu 565 570 575 AsnAsn Tyr Pro Ala Gly Asp Pro Arg Leu Ala Gln Ala Lys Leu Trp 580 585 590Glu Glu Ala Phe Leu Glu Glu Met Arg Ala Phe Gln Arg Arg Met Ala 595 600605 Gly Met Phe Gln Val Thr Phe Thr Ala Glu Arg Ser Leu Glu Asp Glu 610615 620 Ile Asn Arg Thr Thr Ala Glu Asp Leu Pro Ile Phe Ala Thr Ser Tyr625 630 635 640 Ile Val Ile Phe Leu Tyr Ile Ser Leu Ala Leu Gly Ser TyrSer Ser 645 650 655 Trp Ser Arg Val Met Val Asp Ser Lys Ala Thr Leu GlyLeu Gly Gly 660 665 670 Val Ala Val Val Leu Gly Ala Val Met Ala Ala MetGly Phe Phe Ser 675 680 685 Tyr Leu Gly Ile Arg Ser Ser Leu Val Ile LeuGln Val Val Pro Phe 690 695 700 Leu Val Leu Ser Val Gly Ala Asp Asn IlePhe Ile Phe Val Leu Glu 705 710 715 720 Tyr Gln Arg Leu Pro Arg Arg ProGly Glu Pro Arg Glu Val His Ile 725 730 735 Gly Arg Ala Leu Gly Arg ValAla Pro Ser Met Leu Leu Cys Ser Leu 740 745 750 Ser Glu Ala Ile Cys PhePhe Leu Gly Ala Leu Thr Pro Met Pro Ala 755 760 765 Val Arg Thr Phe AlaLeu Thr Ser Gly Leu Ala Val Ile Leu Asp Phe 770 775 780 Leu Leu Gln MetSer Ala Phe Val Ala Leu Leu Ser Leu Asp Ser Lys 785 790 795 800 Arg GlnGlu Ala Ser Arg Leu Asp Val Cys Cys Cys Val Lys Pro Gln 805 810 815 GluLeu Pro Pro Pro Gly Gln Gly Glu Gly Leu Leu Leu Gly Phe Phe 820 825 830Gln Lys Ala Tyr Ala Pro Phe Leu Leu His Trp Ile Thr Arg Gly Val 835 840845 Val Leu Leu Leu Phe Leu Ala Leu Phe Gly Val Ser Leu Tyr Ser Met 850855 860 Cys His Ile Ser Val Gly Leu Asp Gln Glu Leu Ala Leu Pro Lys Asp865 870 875 880 Ser Tyr Leu Leu Asp Tyr Phe Leu Phe Leu Asn Arg Tyr PheGlu Val 885 890 895 Gly Ala Pro Val Tyr Phe Val Thr Thr Leu Gly Tyr AsnPhe Ser Ser 900 905 910 Glu Ala Gly Met Asn Ala Ile Cys Ser Ser Ala GlyCys Asn Asn Phe 915 920 925 Ser Phe Thr Gln Lys Ile Gln Tyr Ala Thr GluPhe Pro Glu Gln Ser 930 935 940 Tyr Leu Ala Ile Pro Ala Ser Ser Trp ValAsp Asp Phe Ile Asp Trp 945 950 955 960 Leu Thr Pro Ser Ser Cys Cys ArgLeu Tyr Ile Ser Gly Pro Asn Lys 965 970 975 Asp Lys Phe Cys Pro Ser ThrVal Asn Ser Leu Asn Cys Leu Lys Asn 980 985 990 Cys Met Ser Ile Thr MetGly Ser Val Arg Pro Ser Val Glu Gln Phe 995 1000 1005 His Lys Tyr LeuPro Trp Phe Leu Asn Asp Arg Pro Asn Ile Lys 1010 1015 1020 Cys Pro LysGly Gly Leu Ala Ala Tyr Ser Thr Ser Val Asn Leu 1025 1030 1035 Thr SerAsp Gly Gln Val Leu Ala Ser Arg Phe Met Ala Tyr His 1040 1045 1050 LysPro Leu Lys Asn Ser Gln Asp Tyr Thr Glu Ala Leu Arg Ala 1055 1060 1065Ala Arg Glu Leu Ala Ala Asn Ile Thr Ala Asp Leu Arg Lys Val 1070 10751080 Pro Gly Thr Asp Pro Ala Phe Glu Val Phe Pro Tyr Thr Ile Thr 10851090 1095 Asn Val Phe Tyr Glu Gln Tyr Leu Thr Ile Leu Pro Glu Gly Leu1100 1105 1110 Phe Met Leu Ser Leu Cys Leu Val Pro Thr Phe Ala Val SerCys 1115 1120 1125 Leu Leu Leu Gly Leu Asp Leu Arg Ser Gly Leu Leu AsnLeu Leu 1130 1135 1140 Ser Ile Val Met Ile Leu Val Asp Thr Val Gly PheMet Ala Leu 1145 1150 1155 Trp Asp Ile Ser Tyr Asn Ala Val Ser Leu IleAsn Leu Val Ser 1160 1165 1170 Ala Val Gly Met Ser Val Glu Phe Val SerHis Ile Thr Arg Ser 1175 1180 1185 Phe Ala Ile Ser Thr Lys Pro Thr TrpLeu Glu Arg Ala Lys Glu 1190 1195 1200 Ala Thr Ile Ser Met Gly Ser AlaVal Phe Ala Gly Val Ala Met 1205 1210 1215 Thr Asn Leu Pro Gly Ile LeuVal Leu Gly Leu Ala Lys Ala Gln 1220 1225 1230 Leu Ile Gln Ile Phe PhePhe Arg Leu Asn Leu Leu Ile Thr Leu 1235 1240 1245 Leu Gly Leu Leu HisGly Leu Val Phe Leu Pro Val Ile Leu Ser 1250 1255 1260 Tyr Val Gly ProAsp Val Asn Pro Ala Leu Ala Leu Glu Gln Lys 1265 1270 1275 Arg Ala GluGlu Ala Val Ala Ala Val Met Val Ala Ser Cys Pro 1280 1285 1290 Asn HisPro Ser Arg Val Ser Thr Ala Asp Asn Ile Tyr Val Asn 1295 1300 1305 HisSer Phe Glu Gly Ser Ile Lys Gly Ala Gly Ala Ile Ser Asn 1310 1315 1320Phe Leu Pro Asn Asn Gly Arg Gln Phe 1325 1330 5 885 DNA Rattus sp. 5ccacgcgtcc gcacctgcaa gtgtggtccc ctgaggcaga gcgcaacatc tccctccagg 60acatctgcta tgcccccctc aacccatata acaccagcct ctccgactgc tgtgtcaaca 120gcctccttca gtacttccag aacaaccgca ccctcctgat gctcacggcc aaccagactc 180tgaatggcca gacctccctg gtggactgga aggaccattt cctctactgt gcaaatgccc 240ctctcacgtt caaagatggc acgtctctgg ccctgagctg catggctgac tacggggctc 300ctgtcttccc cttccttgct gttgggggat accaaggcac ggactattcc gaggcagaag 360cgctgatcat aaccttctct ctcaataact accccgctga tgatccccgc atggcccagg 420ccaagctctg ggaggaggct ttcttgaagg aaatggaatc cttccagagg aacacaagtg 480acaagttcca ggttgcgttc tcagctgagc gctctctgga ggatgagatc aaccgcacca 540ccatccagga cctgcctgtc tttgccgtca gctacattat cgtcttcctg tacatctccc 600tggccctggg cagctactcc agatgcagcc gagtagcggt ggagtccaag gctactctgg 660gcctaggtgg ggtgatagtg tgctgggagc agttctggct tgcatggggc ttctaactcc 720tacctgggtg tcccctcttc tctggttatc atccaagtgg tacctttcct ggtgcttaag 780ctgtgggagc tggacacatc tacatcctag acttgagtac cagaggtacc taggaagccg 840cggaacagcg aaaaggacac attgggcgca ccctgggcat gtggc 885 6 458 DNA Rattussp. 6 gaccagatgt taaccaagct ctggtacagg aggagaaact agccagcgag gcagcagtgg60 ccccagagcc ttcttgccca cagtacccct cccctgctga tgcggatgcc aatgttaact 120acggctttgc cccagaactt gcccacggag ctaatgctgc tagaagctct ttgcccaaaa 180gtgaccaaaa gttctaatgg agtaggagct tgtccatgct tctgctgatg agggatcatg 240aaggtcttcc ctctggttgt cctcaaggcc tggggggagg ttgttcagag aaaaatggct 300ggcattcctg ccacgaggca accggcagct tggcactgac tccttggtct cataggtccc 360taaggcttgg tcagattact cctcatggag agactatctt aagtatctaa gctatcgatt 420gggatgcatc gctgttcatt aaaaaggcta tggctatg 458 7 896 DNA Rattus sp. 7ccacgcgtcc gcagtttcat aagtacctgc cctggttcct gaatgatccg cccaatatca 60gatgtcccaa agggggtcta gcagcgtata gaacgtctgt gaatttgagc tcagatggcc 120aggttatagc ctcccagttc atggcctacc acaagccctt aaggaactca caggacttca 180cagaagctct ccgggcgtcc cggttgctag cagccaacat cacagctgac ctacggaagg 240tgcctgggac agatccaaac tttgaggtct tcccttacac gatctccaac gtgttctacc 300agcaatacct gacggtcctt cctgagggaa tcttcaccct tgctctttgc tttgtgccca 360cctttgttgt ctgctacctc ctactgggcc tggacatgtg ctcagggatc ctcaacctac 420tctccatcat tatgattctc gtggacacca ttggcctcat ggctgtgtgg ggtatcagct 480ataatgcggt atccctcatc aaccttgtca cggcagtggg catgtctgtg gagtttgtgt 540cccacatcac tcggtccttt gcttgtaagc accaagccta cccggctgga gagggctaaa 600agatgctact gtcttcatgg gcagtgcggt gtttgctgga gtggccatga ccaacttccc 660aggcatcctc atcttggggg ctttgcccca agcccaggct tattcagatc ttcttcttcc 720gcctcaacct tctgatcacc tttgctgggg tctgctgcat ggctggtctt cctgcccggt 780ttgtcctcag ctatctggga ccagatgtaa ccaaggctct gctacccgga ggagaaacta 840gccagcgagg gcagcagtgg ccccagagac ttcttgccca caagtaccct tccctg 896 8 3124DNA Rattus sp. 8 tgcaagtgtg gtcccctgag gcagagcgca acatctccct ccaggacatctgctatgccc 60 ccctcaaccc atataacacc agcctctccg actgctgtgt caacagcctccttcagtact 120 tccagaacaa ccgcaccctc ctgatgctca cggccaacca gactctgaatggccagacct 180 ccctggtgga ctggaaggac catttcctct actgtgcaaa tgcccctctcacgttcaaag 240 atggcacgtc tctggccctg agctgcatgg ctgactacgg ggctcctgtcttccccttcc 300 ttgctgttgg gggataccaa ggcacggact attccgaggc agaagcgctgatcataacct 360 tctctctcaa taactacccc gctgatgatc cccgcatggc ccaggccaagctctgggagg 420 aggctttctt gaaggaaatg gaatccttcc agaggaacac aagtgacaagttccaggttg 480 cgttctcagc tgagcgctct ctggaggatg agatcaaccg caccaccatccaggacctgc 540 ctgtctttgc cgtcagctac attatcgtct tcctgtacat ctccctggccctgggcagct 600 actccagatg cagccgagta gcggtggagt ccaaggctac tctgggcctaggtggggtga 660 ttgttgtgct gggagcagtt ctggctgcca tgggcttcta ctcctacctgggtgtcccct 720 cttctctggt tatcatccaa gtggtacctt tcctggtgct agctgtgggagctgacaaca 780 tcttcatctt tgttcttgag taccagaggc tacctaggat gcctggggaacagcgagagg 840 ctcacattgg ccgcaccctg ggcagtgtgg cccccagcat gctgctgtgcagcctctctg 900 aggccatctg cttctttcta ggggccctga cccccatgcc agctgtgaggaccttcgcct 960 tgacctctgg cttagcaatt atcctcgact tcctgctcca gatgactgcctttgtggccc 1020 tgctctccct ggatagcaag aggcaggagg cctctcgccc ggatgtcttatgctgctttt 1080 caacccggaa gctgccccca cctaaagaaa aagaaggcct cttactccgcttcttccgca 1140 agatatacgc tcctttcctg ctgcacagat tcatccgccc tgttgtgatgctgctgtttc 1200 tgaccctgtt tggagcaaat ctctacttaa tgtgcaacat caacgtggggctagaccagg 1260 agctggctct gcccaaggac tcgtacttga tagactactt cctctttctgaaccgatacc 1320 ttgaagtggg gcctccagtg tactttgtca ccacctcggg cttcaacttctccagcgagg 1380 caggcatgaa cgccacttgc tctagcgcag gctgtaagag cttctccctaacccagaaaa 1440 tccagtatgc cagtgaattc cctgaccagt cttacgtggc tattgctgcatcctcctggg 1500 tagatgactt catcgactgg ctgaccccgt cctcctcctg ctgtcgcctttatatacgtg 1560 gcccccataa ggatgagttc tgtccctcaa cggatacttc cttcaactgcttaaaaaact 1620 gcatgaaccg cactctgggt cctgtgaggc ccacagcgga acagtttcataagtacctgc 1680 cctggttcct gaatgatccg cccaatatca gatgtcccaa agggggtctagcagcgtata 1740 gaacgtctgt gaatttgagc tcagatggcc aggttatagc ctcccagttcatggcctacc 1800 acaagccctt aaggaactca caggacttca cagaagctct ccgggcgtcccggttgctag 1860 cagccaacat cacagctgac ctacggaagg tgcctgggac agatccaaactttgaggtct 1920 tcccttacac gatctccaac gtgttctacc agcaatacct gacggtccttcctgagggaa 1980 tcttcaccct tgctctttgc tttgtgccca cctttgttgt ctgctacctcctactgggcc 2040 tggacatgtg ctcagggatc ctcaacctac tctccatcat tatgattctcgtggacacca 2100 ttggcctcat ggctgtgtgg ggtatcagct ataatgcggt atccctcatcaaccttgtca 2160 cggcagtggg catgtctgtg gagtttgtgt cccacatcac tcggtcctttgctgtaagca 2220 ccaagcctac ccggctggag agggctaaag atgctactgt cttcatgggcagtgcggtgt 2280 ttgctggagt ggccatgacc aacttcccag gcatcctcat cttgggctttgcccaagccc 2340 agcttattca gatcttcttc ttccgcctca accttctgat caccttgctgggtctgctgc 2400 atggcctggt cttcctgccg gttgtcctca gctatctggg accagatgttaaccaagctc 2460 tggtacagga ggagaaacta gccagcgagg cagcagtggc cccagagccttcttgcccac 2520 agtacccctc ccctgctgat gcggatgcca atgttaacta cggctttgccccagaacttg 2580 cccacggagc taatgctgct agaagctctt tgcccaaaag tgaccaaaagttctaatgga 2640 gtaggagctt gtccatgctt cttgctgatg agggatcatg aaggtcttccctctggttgt 2700 cctcaaggcc tggggggagg ttgtttcaga gaaaaatggc tggcattcctgccacgaggc 2760 aaccggcagc attggcactg acctccttgc tctcataggt ccctaaggccttggtcagat 2820 tacctcctcc atggagagac tatcttaagt atcttaagta tcgtatgggatgcatcgcct 2880 gtcaattaaa aaggctatgg cctatggctc aggcagggcc atccggaagaagagaggatt 2940 ctgggataaa gccaggtggg agattcgcct ggggaaaatg tgacaatggttcctgagcat 3000 gggcaatcag ccatgtggca gaatgtaaat taatataaat gggttgtcttaagttatgat 3060 tctagctggg gaggagccta gctgtgtagc caagatattt gtaaatataaaaaaaaaaaa 3120 aaaa 3124 9 4484 DNA Rattus sp. 9 atggcagctg cctggctgggatggctgctc tgggccctgc tcctgagcgc ggcccagggt 60 gagctataca cacccaaacacgaagctggg gtctgcacct tttacgaaga gtgcgggaaa 120 aacccagagc tctctggaggcctcacgtca ctatccaatg tatcctgcct gtctaacacc 180 ccggcccgcc acgtcacgggtgaacacctg gctcttctcc agcgcatctg tccccgcctg 240 tacaacggcc ccaataccacttttgcctgt tgctctacca agcagctgct gtccttagaa 300 agcagcatgt ccatcaccaaggcccttctc acgcgctgcc cggcctgctc tgacaatttt 360 gtgagcttac actgccacaacacttgcagc cctgaccaga gcctcttcat caacgtcacc 420 cgggtggttg agcggggcgctggagagcct cctgccgtgg tggcctatga ggccttttat 480 cagcgcagct ttgctgagaaggcctatgag tcctgcagcc aggtgcgcat ccctgcggcc 540 gcttccttgg ccgtgggcagcatgtgtgga gtgtatggct ccgccctctg caatgctcag 600 cgctggctca acttccaaggagacacaggg aatggcctgg ctccgctgga tatcaccttc 660 cacctcttgg agcctggccaggccctaccg gatgggatcc agccactgaa tgggaagatc 720 gcaccctgca acgagtctcagggtgatgac tcagcagtct gctcctgcca ggactgtgcg 780 gcgtcctgcc ctgtcatccctccgcccgag gccttgcgcc cttccttcta catgggtcgc 840 atgccaggct ggctggccctcatcatcatc ttcactgctg tctttgtgtt gctctctgca 900 gtccttgtgc gtctccgagtggtttccaac aggaacaaga acaaggcaga aggcccccag 960 gaagccccca aactccctcataagcacaaa ctctcacccc ataccatcct gggccggttc 1020 ttccagaact ggggcacaagggtggcctcg tggccactca ccgtcttagc actgtccttc 1080 atcgttgtga tagccttagcagcaggcctg acctttattg aactcaccac agaccctgtg 1140 gaactgtggt cggcccccaagagccaggcc cggaaagaga agtctttcca tgatgagcat 1200 ttcggcccct tctttcgaaccaaccagatt ttcgtgacag ctcggaacag gtccagctac 1260 aagtacgact ccctactgctagggtccaag aacttcagtg ggatcctgtc cctggacttc 1320 ctgctggagc tgctggagcttcaggagagg cttcgacacc tgcaagtgtg gtcccctgag 1380 gcagagcgca acatctccctccaggacatc tgctatgccc ccctcaaccc atataacacc 1440 agcctctccg actgctgtgtcaacagcctc cttcagtact tccagaacaa ccgcaccctc 1500 ctgatgctca cggccaaccagactctgaat ggccagacct ccctggtgga ctggaaggac 1560 catttcctct actgtgcaaatgcccctctc acgttcaaag atggcacgtc tctggccctg 1620 agctgcatgg ctgactacggggctcctgtc ttccccttcc ttgctgttgg gggataccaa 1680 ggcacggact attccgaggcagaagcgctg atcataacct tctctctcaa taactacccc 1740 gctgatgatc cccgcatggcccaggccaag ctctgggagg aggctttctt gaaggaaatg 1800 gaatccttcc agaggaacacaagtgacaag ttccaggttg cgttctcagc tgagcgctct 1860 ctggaggatg agatcaaccgcaccaccatc caggacctgc ctgtctttgc cgtcagctac 1920 attatcgtct tcctgtacatctccctggcc ctgggcagct actccagatg cagccgagta 1980 gcggtggagt ccaaggctactctgggccta ggtggggtga ttgttgtgct gggagcagtt 2040 ctggctgcca tgggcttctactcctacctg ggtgtcccct cttctctggt tatcatccaa 2100 gtggtacctt tcctggtgctagctgtggga gctgacaaca tcttcatctt tgttcttgag 2160 taccagaggc tacctaggatgcctggggaa cagcgagagg ctcacattgg ccgcaccctg 2220 ggcagtgtgg cccccagcatgctgctgtgc agcctctctg aggccatctg cttctttcta 2280 ggggccctga cccccatgccagctgtgagg accttcgcct tgacctctgg cttagcaatt 2340 atcctcgact tcctgctccagatgactgcc tttgtggccc tgctctccct ggatagcaag 2400 aggcaggagg cctctcgcccggatgtctta tgctgctttt caacccggaa gctgccccca 2460 cctaaagaaa aagaaggcctcttactccgc ttcttccgca agatatacgc tcctttcctg 2520 ctgcacagat tcatccgccctgttgtgatg ctgctgtttc tgaccctgtt tggagcaaat 2580 ctctacttaa tgtgcaacatcaacgtgggg ctagaccagg agctggctct gcccaaggac 2640 tcgtacttga tagactacttcctctttctg aaccgatacc ttgaagtggg gcctccagtg 2700 tactttgtca ccacctcgggcttcaacttc tccagcgagg caggcatgaa cgccacttgc 2760 tctagcgcag gctgtaagagcttctcccta acccagaaaa tccagtatgc cagtgaattc 2820 cctgaccagt cttacgtggctattgctgca tcctcctggg tagatgactt catcgactgg 2880 ctgaccccgt cctcctcctgctgtcgcctt tatatacgtg gcccccataa ggatgagttc 2940 tgtccctcaa cggatacttccttcaactgc ttaaaaaact gcatgaaccg cactctgggt 3000 cctgtgaggc ccacagcggaacagtttcat aagtacctgc cctggttcct gaatgatccg 3060 cccaatatca gatgtcccaaagggggtcta gcagcgtata gaacgtctgt gaatttgagc 3120 tcagatggcc aggttatagcctcccagttc atggcctacc acaagccctt aaggaactca 3180 caggacttca cagaagctctccgggcgtcc cggttgctag cagccaacat cacagctgac 3240 ctacggaagg tgcctgggacagatccaaac tttgaggtct tcccttacac gatctccaac 3300 gtgttctacc agcaatacctgacggtcctt cctgagggaa tcttcaccct tgctctttgc 3360 tttgtgccca cctttgttgtctgctacctc ctactgggcc tggacatgtg ctcagggatc 3420 ctcaacctac tctccatcattatgattctc gtggacacca ttggcctcat ggctgtgtgg 3480 ggtatcagct ataatgcggtatccctcatc aaccttgtca cggcagtggg catgtctgtg 3540 gagtttgtgt cccacatcactcggtccttt gctgtaagca ccaagcctac ccggctggag 3600 agggctaaag atgctactgtcttcatgggc agtgcggtgt ttgctggagt ggccatgacc 3660 aacttcccag gcatcctcatcttgggcttt gcccaagccc agcttattca gatcttcttc 3720 ttccgcctca accttctgatcaccttgctg ggtctgctgc atggcctggt cttcctgccg 3780 gttgtcctca gctatctgggaccagatgtt aaccaagctc tggtacagga ggagaaacta 3840 gccagcgagg cagcagtggccccagagcct tcttgcccac agtacccctc ccctgctgat 3900 gcggatgcca atgttaactacggctttgcc ccagaacttg cccacggagc taatgctgct 3960 agaagctctt tgcccaaaagtgaccaaaag ttctaatgga gtaggagctt gtccatgctt 4020 cttgctgatg agggatcatgaaggtcttcc ctctggttgt cctcaaggcc tggggggagg 4080 ttgtttcaga gaaaaatggctggcattcct gccacgaggc aaccggcagc attggcactg 4140 acctccttgc tctcataggtccctaaggcc ttggtcagat tacctcctcc atggagagac 4200 tatcttaagt atcttaagtatcgtatggga tgcatcgcct gtcaattaaa aaggctatgg 4260 cctatggctc aggcagggccatccggaaga agagaggatt ctgggataaa gccaggtggg 4320 agattcgcct ggggaaaatgtgacaatggt tcctgagcat gggcaatcag ccatgtggca 4380 gaatgtaaat taatataaatgggttgtctt aagttatgat tctagctggg gaggagccta 4440 gctgtgtagc caagatatttgtaaatataa aaaaaaaaaa aaaa 4484 10 3993 DNA Rattus sp. misc_feature(1)..(3993) n is g or a or t or c 10 atggcngcng cntggytngg ntggytnytntgggcnytny tnytnwsngc ngcncarggn 60 garytntaya cnccnaarca ygargcnggngtntgyacnt tytaygarga rtgyggnaar 120 aayccngary tnwsnggngg nytnacnwsnytnwsnaayg tnwsntgyyt nwsnaayacn 180 ccngcnmgnc aygtnacngg ngarcayytngcnytnytnc armgnathtg yccnmgnytn 240 tayaayggnc cnaayacnac nttygcntgytgywsnacna arcarytnyt nwsnytngar 300 wsnwsnatgw snathacnaa rgcnytnytnacnmgntgyc cngcntgyws ngayaaytty 360 gtnwsnytnc aytgycayaa yacntgywsnccngaycarw snytnttyat haaygtnacn 420 mgngtngtng armgnggngc nggngarccnccngcngtng tngcntayga rgcnttytay 480 carmgnwsnt tygcngaraa rgcntaygarwsntgywsnc argtnmgnat hccngcngcn 540 gcnwsnytng cngtnggnws natgtgyggngtntayggnw sngcnytntg yaaygcncar 600 mgntggytna ayttycargg ngayacnggnaayggnytng cnccnytnga yathacntty 660 cayytnytng arccnggnca rgcnytnccngayggnathc arccnytnaa yggnaarath 720 gcnccntgya aygarwsnca rggngaygaywsngcngtnt gywsntgyca rgaytgygcn 780 gcnwsntgyc cngtnathcc nccnccngargcnytnmgnc cnwsnttyta yatgggnmgn 840 atgccnggnt ggytngcnyt nathathathttyacngcng tnttygtnyt nytnwsngcn 900 gtnytngtnm gnytnmgngt ngtnwsnaaymgnaayaara ayaargcnga rggnccncar 960 gargcnccna arytnccnca yaarcayaarytnwsnccnc ayacnathyt nggnmgntty 1020 ttycaraayt ggggnacnmg ngtngcnwsntggccnytna cngtnytngc nytnwsntty 1080 athgtngtna thgcnytngc ngcnggnytnacnttyathg arytnacnac ngayccngtn 1140 garytntggw sngcnccnaa rwsncargcnmgnaargara arwsnttyca ygaygarcay 1200 ttyggnccnt tyttymgnac naaycarathttygtnacng cnmgnaaymg nwsnwsntay 1260 aartaygayw snytnytnyt nggnwsnaaraayttywsng gnathytnws nytngaytty 1320 ytnytngary tnytngaryt ncargarmgnytnmgncayy tncargtntg gwsnccngar 1380 gcngarmgna ayathwsnyt ncargayathtgytaygcnc cnytnaaycc ntayaayacn 1440 wsnytnwsng aytgytgygt naaywsnytnytncartayt tycaraayaa ymgnacnytn 1500 ytnatgytna cngcnaayca racnytnaayggncaracnw snytngtnga ytggaargay 1560 cayttyytnt aytgygcnaa ygcnccnytnacnttyaarg ayggnacnws nytngcnytn 1620 wsntgyatgg cngaytaygg ngcnccngtnttyccnttyy tngcngtngg nggntaycar 1680 ggnacngayt aywsngargc ngargcnytnathathacnt tywsnytnaa yaaytayccn 1740 gcngaygayc cnmgnatggc ncargcnaarytntgggarg argcnttyyt naargaratg 1800 garwsnttyc armgnaayac nwsngayaarttycargtng cnttywsngc ngarmgnwsn 1860 ytngargayg arathaaymg nacnacnathcargayytnc cngtnttygc ngtnwsntay 1920 athathgtnt tyytntayat hwsnytngcnytnggnwsnt aywsnmgntg ywsnmgngtn 1980 gcngtngarw snaargcnac nytnggnytnggnggngtna thgtngtnyt nggngcngtn 2040 ytngcngcna tgggnttyta ywsntayytnggngtnccnw snwsnytngt nathathcar 2100 gtngtnccnt tyytngtnyt ngcngtnggngcngayaaya thttyathtt ygtnytngar 2160 taycarmgny tnccnmgnat gccnggngarcarmgngarg cncayathgg nmgnacnytn 2220 ggnwsngtng cnccnwsnat gytnytntgywsnytnwsng argcnathtg yttyttyytn 2280 ggngcnytna cnccnatgcc ngcngtnmgnacnttygcny tnacnwsngg nytngcnath 2340 athytngayt tyytnytnca ratgacngcnttygtngcny tnytnwsnyt ngaywsnaar 2400 mgncargarg cnwsnmgncc ngaygtnytntgytgyttyw snacnmgnaa rytnccnccn 2460 ccnaargara argarggnyt nytnytnmgnttyttymgna arathtaygc nccnttyytn 2520 ytncaymgnt tyathmgncc ngtngtnatgytnytnttyy tnacnytntt yggngcnaay 2580 ytntayytna tgtgyaayat haaygtnggnytngaycarg arytngcnyt nccnaargay 2640 wsntayytna thgaytaytt yytnttyytnaaymgntayy tngargtngg nccnccngtn 2700 tayttygtna cnacnwsngg nttyaayttywsnwsngarg cnggnatgaa ygcnacntgy 2760 wsnwsngcng gntgyaarws nttywsnytnacncaraara thcartaygc nwsngartty 2820 ccngaycarw sntaygtngc nathgcngcnwsnwsntggg tngaygaytt yathgaytgg 2880 ytnacnccnw snwsnwsntg ytgymgnytntayathmgng gnccncayaa rgaygartty 2940 tgyccnwsna cngayacnws nttyaaytgyytnaaraayt gyatgaaymg nacnytnggn 3000 ccngtnmgnc cnacngcnga rcarttycayaartayytnc cntggttyyt naaygayccn 3060 ccnaayathm gntgyccnaa rggnggnytngcngcntaym gnacnwsngt naayytnwsn 3120 wsngayggnc argtnathgc nwsncarttyatggcntayc ayaarccnyt nmgnaaywsn 3180 cargayttya cngargcnyt nmgngcnwsnmgnytnytng cngcnaayat hacngcngay 3240 ytnmgnaarg tnccnggnac ngayccnaayttygargtnt tyccntayac nathwsnaay 3300 gtnttytayc arcartayyt nacngtnytnccngarggna thttyacnyt ngcnytntgy 3360 ttygtnccna cnttygtngt ntgytayytnytnytnggny tngayatgtg ywsnggnath 3420 ytnaayytny tnwsnathat hatgathytngtngayacna thggnytnat ggcngtntgg 3480 ggnathwsnt ayaaygcngt nwsnytnathaayytngtna cngcngtngg natgwsngtn 3540 garttygtnw sncayathac nmgnwsnttygcngtnwsna cnaarccnac nmgnytngar 3600 mgngcnaarg aygcnacngt nttyatgggnwsngcngtnt tygcnggngt ngcnatgacn 3660 aayttyccng gnathytnat hytnggnttygcncargcnc arytnathca rathttytty 3720 ttymgnytna ayytnytnat hacnytnytnggnytnytnc ayggnytngt nttyytnccn 3780 gtngtnytnw sntayytngg nccngaygtnaaycargcny tngtncarga rgaraarytn 3840 gcnwsngarg cngcngtngc nccngarccnwsntgyccnc artayccnws nccngcngay 3900 gcngaygcna aygtnaayta yggnttygcnccngarytng cncayggngc naaygcngcn 3960 mgnwsnwsny tnccnaarws ngaycaraartty 3993 11 4002 DNA Mus sp. CDS (1)..(4002) 11 atg gca gct gcc tgg caggga tgg ctg ctc tgg gcc ctg ctc ctg aat 48 Met Ala Ala Ala Trp Gln GlyTrp Leu Leu Trp Ala Leu Leu Leu Asn 1 5 10 15 tcg gcc cag ggt gag ctctac aca ccc act cac aaa gct ggc ttc tgc 96 Ser Ala Gln Gly Glu Leu TyrThr Pro Thr His Lys Ala Gly Phe Cys 20 25 30 acc ttt tat gaa gag tgt gggaag aac cca gag ctt tct gga ggc ctc 144 Thr Phe Tyr Glu Glu Cys Gly LysAsn Pro Glu Leu Ser Gly Gly Leu 35 40 45 aca tca cta tcc aat atc tcc tgcttg tct aat acc cca gcc cgc cat 192 Thr Ser Leu Ser Asn Ile Ser Cys LeuSer Asn Thr Pro Ala Arg His 50 55 60 gtc aca ggt gac cac ctg gct ctt ctccag cgc gtc tgt ccc cgc cta 240 Val Thr Gly Asp His Leu Ala Leu Leu GlnArg Val Cys Pro Arg Leu 65 70 75 80 tac aat ggc ccc aat gac acc tat gcctgt tgc tct acc aag cag ctg 288 Tyr Asn Gly Pro Asn Asp Thr Tyr Ala CysCys Ser Thr Lys Gln Leu 85 90 95 gtg tca tta gac agt agc ctg tct atc accaag gcc ctc ctt aca cgc 336 Val Ser Leu Asp Ser Ser Leu Ser Ile Thr LysAla Leu Leu Thr Arg 100 105 110 tgc ccg gca tgc tct gaa aat ttt gtg agcata cac tgt cat aat acc 384 Cys Pro Ala Cys Ser Glu Asn Phe Val Ser IleHis Cys His Asn Thr 115 120 125 tgc agc cct gac cag agc ctc ttc atc aatgtt act cgc gtg gtt cag 432 Cys Ser Pro Asp Gln Ser Leu Phe Ile Asn ValThr Arg Val Val Gln 130 135 140 cgg gac cct gga cag ctt cct gct gtg gtggcc tat gag gcc ttt tat 480 Arg Asp Pro Gly Gln Leu Pro Ala Val Val AlaTyr Glu Ala Phe Tyr 145 150 155 160 caa cgc agt ttt gca gag aag gcc tatgag tcc tgt agc cgg gtg cgc 528 Gln Arg Ser Phe Ala Glu Lys Ala Tyr GluSer Cys Ser Arg Val Arg 165 170 175 atc cct gca gct gcc tcg ctg gct gtgggc agc atg tgt gga gtg tat 576 Ile Pro Ala Ala Ala Ser Leu Ala Val GlySer Met Cys Gly Val Tyr 180 185 190 ggc tct gcc ctc tgc aat gct cag cgctgg ctc aac ttc caa gga gac 624 Gly Ser Ala Leu Cys Asn Ala Gln Arg TrpLeu Asn Phe Gln Gly Asp 195 200 205 aca ggg aat ggc ctg gct ccg ctg gacatc acc ttc cac ctc ttg gag 672 Thr Gly Asn Gly Leu Ala Pro Leu Asp IleThr Phe His Leu Leu Glu 210 215 220 cct ggc cag gcc ctg gca gat ggg atgaag cca ctg gat ggg aag atc 720 Pro Gly Gln Ala Leu Ala Asp Gly Met LysPro Leu Asp Gly Lys Ile 225 230 235 240 aca ccc tgc aat gag tcc cag ggtgaa gac tcg gca gcc tgt tcc tgc 768 Thr Pro Cys Asn Glu Ser Gln Gly GluAsp Ser Ala Ala Cys Ser Cys 245 250 255 cag gac tgt gca gca tcc tgc cctgtc atc cct ccg ccc ccg gcc ctg 816 Gln Asp Cys Ala Ala Ser Cys Pro ValIle Pro Pro Pro Pro Ala Leu 260 265 270 cgc cct tct ttc tac atg ggt cgaatg cca ggc tgg ctg gct ctc atc 864 Arg Pro Ser Phe Tyr Met Gly Arg MetPro Gly Trp Leu Ala Leu Ile 275 280 285 atc atc ttc act gct gtc ttt gtattg ctc tct gtt gtc ctt gtg tat 912 Ile Ile Phe Thr Ala Val Phe Val LeuLeu Ser Val Val Leu Val Tyr 290 295 300 ctc cga gtg gct tcc aac agg aacaag aac aag aca gca ggc tcc cag 960 Leu Arg Val Ala Ser Asn Arg Asn LysAsn Lys Thr Ala Gly Ser Gln 305 310 315 320 gaa gcc ccc aac ctc cct cgtaag cgc aga ttc tca cct cac act gtc 1008 Glu Ala Pro Asn Leu Pro Arg LysArg Arg Phe Ser Pro His Thr Val 325 330 335 ctt ggc cgg ttc ttc gag agctgg gga aca agg gtg gcc tca tgg cca 1056 Leu Gly Arg Phe Phe Glu Ser TrpGly Thr Arg Val Ala Ser Trp Pro 340 345 350 ctc act gtc ttg gca ctg tccttc ata gtt gtg ata gcc ttg tca gta 1104 Leu Thr Val Leu Ala Leu Ser PheIle Val Val Ile Ala Leu Ser Val 355 360 365 ggc ctg acc ttt ata gaa ctcacc aca gac cct gtg gaa ctg tgg tcg 1152 Gly Leu Thr Phe Ile Glu Leu ThrThr Asp Pro Val Glu Leu Trp Ser 370 375 380 gcc cct aaa agc caa gcc cggaaa gaa aag gct ttc cat gac gag cat 1200 Ala Pro Lys Ser Gln Ala Arg LysGlu Lys Ala Phe His Asp Glu His 385 390 395 400 ttt ggc ccc ttc ttc cgaacc aac cag att ttt gtg aca gct aag aac 1248 Phe Gly Pro Phe Phe Arg ThrAsn Gln Ile Phe Val Thr Ala Lys Asn 405 410 415 agg tcc agc tac aag tacgac tcc ctg ctg cta ggg ccc aag aac ttc 1296 Arg Ser Ser Tyr Lys Tyr AspSer Leu Leu Leu Gly Pro Lys Asn Phe 420 425 430 agt ggg atc cta tcc ctggac ttg ctg cag gag ctg ttg gag cta cag 1344 Ser Gly Ile Leu Ser Leu AspLeu Leu Gln Glu Leu Leu Glu Leu Gln 435 440 445 gag aga ctt cga cac ctgcaa gtg tgg tcc cat gag gca cag cgc aac 1392 Glu Arg Leu Arg His Leu GlnVal Trp Ser His Glu Ala Gln Arg Asn 450 455 460 atc tcc ctc cag gac atctgc tat gct ccc ctc aac ccg cat aac acc 1440 Ile Ser Leu Gln Asp Ile CysTyr Ala Pro Leu Asn Pro His Asn Thr 465 470 475 480 agc ctc act gac tgctgt gtc aac agc ctc ctt caa tac ttc cag aac 1488 Ser Leu Thr Asp Cys CysVal Asn Ser Leu Leu Gln Tyr Phe Gln Asn 485 490 495 aac cac aca ctc ctgctg ctc aca gcc aat cag act ctg aat ggc cag 1536 Asn His Thr Leu Leu LeuLeu Thr Ala Asn Gln Thr Leu Asn Gly Gln 500 505 510 acc tcc ctg gtg gactgg aag gac cat ttc ctc tac tgt gcc aat gcc 1584 Thr Ser Leu Val Asp TrpLys Asp His Phe Leu Tyr Cys Ala Asn Ala 515 520 525 cct ctc acg tac aaagat ggc aca gcc ctg gcc ctg agc tgc ata gct 1632 Pro Leu Thr Tyr Lys AspGly Thr Ala Leu Ala Leu Ser Cys Ile Ala 530 535 540 gac tac ggg gca cctgtc ttc ccc ttc ctt gct gtt ggg ggc tac caa 1680 Asp Tyr Gly Ala Pro ValPhe Pro Phe Leu Ala Val Gly Gly Tyr Gln 545 550 555 560 ggg acg gac tactcg gag gca gaa gcc ctg atc ata acc ttc tct atc 1728 Gly Thr Asp Tyr SerGlu Ala Glu Ala Leu Ile Ile Thr Phe Ser Ile 565 570 575 aat aac tac cccgct gat gat ccc cgc atg gcc cac gcc aag ctc tgg 1776 Asn Asn Tyr Pro AlaAsp Asp Pro Arg Met Ala His Ala Lys Leu Trp 580 585 590 gag gag gct ttcttg aag gaa atg caa tcc ttc cag aga agc aca gct 1824 Glu Glu Ala Phe LeuLys Glu Met Gln Ser Phe Gln Arg Ser Thr Ala 595 600 605 gac aag ttc cagatt gcg ttc tca gct gag cgt tct ctg gag gac gag 1872 Asp Lys Phe Gln IleAla Phe Ser Ala Glu Arg Ser Leu Glu Asp Glu 610 615 620 atc aat cgc actacc atc cag gac ctg cct gtc ttt gcc atc agc tac 1920 Ile Asn Arg Thr ThrIle Gln Asp Leu Pro Val Phe Ala Ile Ser Tyr 625 630 635 640 ctt atc gtcttc ctg tac atc tcc ctg gcc ctg ggc agc tac tcc aga 1968 Leu Ile Val PheLeu Tyr Ile Ser Leu Ala Leu Gly Ser Tyr Ser Arg 645 650 655 tgg agc cgagtt gcg gtg gat tcc aag gct act ctg ggc cta ggt ggg 2016 Trp Ser Arg ValAla Val Asp Ser Lys Ala Thr Leu Gly Leu Gly Gly 660 665 670 gtg gct gttgtg ctg gga gca gtc gtc gct gcc atg ggc ttc tac tcc 2064 Val Ala Val ValLeu Gly Ala Val Val Ala Ala Met Gly Phe Tyr Ser 675 680 685 tac ctg ggtgtc ccc tcc tct ctg gtc atc att caa gtg gta cct ttc 2112 Tyr Leu Gly ValPro Ser Ser Leu Val Ile Ile Gln Val Val Pro Phe 690 695 700 ctg gtg ctggct gtg gga gct gac aac atc ttc atc ttt gtt ctt gag 2160 Leu Val Leu AlaVal Gly Ala Asp Asn Ile Phe Ile Phe Val Leu Glu 705 710 715 720 tac cagagg ctg cct agg atg ccc ggg gag cag cga gag gct cac att 2208 Tyr Gln ArgLeu Pro Arg Met Pro Gly Glu Gln Arg Glu Ala His Ile 725 730 735 ggc cgcacc ctg ggt agt gtg gcc ccc agc atg ctg ctg tgc agc ctc 2256 Gly Arg ThrLeu Gly Ser Val Ala Pro Ser Met Leu Leu Cys Ser Leu 740 745 750 tct gaggcc atc tgc ttc ttt cta ggg gcc ctg acc tcc atg cca gct 2304 Ser Glu AlaIle Cys Phe Phe Leu Gly Ala Leu Thr Ser Met Pro Ala 755 760 765 gtg aggacc ttt gcc ttg acc tct ggc tta gca atc atc ttt gac ttc 2352 Val Arg ThrPhe Ala Leu Thr Ser Gly Leu Ala Ile Ile Phe Asp Phe 770 775 780 ctg ctccag atg aca gcc ttt gtg gcc ctg ctc tcc ctg gat agc aag 2400 Leu Leu GlnMet Thr Ala Phe Val Ala Leu Leu Ser Leu Asp Ser Lys 785 790 795 800 aggcag gag gcc tct cgc ccc gac gtc gtg tgc tgc ttt tca agc cga 2448 Arg GlnGlu Ala Ser Arg Pro Asp Val Val Cys Cys Phe Ser Ser Arg 805 810 815 aatctg ccc cca ccg aaa caa aaa gaa ggc ctc tta ctt tgc ttc ttc 2496 Asn LeuPro Pro Pro Lys Gln Lys Glu Gly Leu Leu Leu Cys Phe Phe 820 825 830 cgcaag ata tac act ccc ttc ctg ctg cac aga ttc atc cgc cct gtt 2544 Arg LysIle Tyr Thr Pro Phe Leu Leu His Arg Phe Ile Arg Pro Val 835 840 845 gtgctg ctg ctc ttt ctg gtc ctg ttt gga gca aac ctc tac tta atg 2592 Val LeuLeu Leu Phe Leu Val Leu Phe Gly Ala Asn Leu Tyr Leu Met 850 855 860 tgcaac atc agc gtg ggg ctg gac cag gat ctg gct ctg ccc aag gat 2640 Cys AsnIle Ser Val Gly Leu Asp Gln Asp Leu Ala Leu Pro Lys Asp 865 870 875 880tcc tac ctg ata gac tac ttc ctc ttt ctg aac cgg tac ttg gaa gtg 2688 SerTyr Leu Ile Asp Tyr Phe Leu Phe Leu Asn Arg Tyr Leu Glu Val 885 890 895ggg cct cca gtg tac ttt gac acc acc tca ggc tac aac ttt tcc acc 2736 GlyPro Pro Val Tyr Phe Asp Thr Thr Ser Gly Tyr Asn Phe Ser Thr 900 905 910gag gca ggc atg aac gcc att tgc tct agt gca ggc tgt gag agc ttc 2784 GluAla Gly Met Asn Ala Ile Cys Ser Ser Ala Gly Cys Glu Ser Phe 915 920 925tcc cta acc cag aaa atc cag tat gcc agt gaa ttc cct aat cag tct 2832 SerLeu Thr Gln Lys Ile Gln Tyr Ala Ser Glu Phe Pro Asn Gln Ser 930 935 940tat gtg gct att gct gca tcc tcc tgg gta gat gac ttc atc gac tgg 2880 TyrVal Ala Ile Ala Ala Ser Ser Trp Val Asp Asp Phe Ile Asp Trp 945 950 955960 ctg acc cca tcc tcc tcc tgc tgc cgc att tat acc cgt ggc ccc cat 2928Leu Thr Pro Ser Ser Ser Cys Cys Arg Ile Tyr Thr Arg Gly Pro His 965 970975 aaa gat gag ttc tgt ccc tca acg gat act tcc ttc aac tgt ctc aaa 2976Lys Asp Glu Phe Cys Pro Ser Thr Asp Thr Ser Phe Asn Cys Leu Lys 980 985990 aac tgc atg aac cgc act ctg ggt ccc gtg aga ccc aca aca gaa cag 3024Asn Cys Met Asn Arg Thr Leu Gly Pro Val Arg Pro Thr Thr Glu Gln 995 10001005 ttt cat aag tac ctg ccc tgg ttc ctg aat gat acg ccc aac atc 3069Phe His Lys Tyr Leu Pro Trp Phe Leu Asn Asp Thr Pro Asn Ile 1010 10151020 aga tgt cct aaa ggg ggc cta gca gcg tat aga acc tct gtg aat 3114Arg Cys Pro Lys Gly Gly Leu Ala Ala Tyr Arg Thr Ser Val Asn 1025 10301035 ttg agc tca gat ggc cag att ata gcc tcc cag ttc atg gcc tac 3159Leu Ser Ser Asp Gly Gln Ile Ile Ala Ser Gln Phe Met Ala Tyr 1040 10451050 cac aag ccc tta cgg aac tca cag gac ttt aca gaa gct ctc cgg 3204His Lys Pro Leu Arg Asn Ser Gln Asp Phe Thr Glu Ala Leu Arg 1055 10601065 gca tcc cgg ttg cta gca gcc aac atc aca gct gaa cta cgg aag 3249Ala Ser Arg Leu Leu Ala Ala Asn Ile Thr Ala Glu Leu Arg Lys 1070 10751080 gtg cct ggg aca gat ccc aac ttt gag gtc ttc cct tac acg atc 3294Val Pro Gly Thr Asp Pro Asn Phe Glu Val Phe Pro Tyr Thr Ile 1085 10901095 tcc aat gtg ttc tac cag caa tac ctg acg gtt ctc cct gag gga 3339Ser Asn Val Phe Tyr Gln Gln Tyr Leu Thr Val Leu Pro Glu Gly 1100 11051110 atc ttc act ctt gct ctc tgc ttc gtg ccc acc ttt gtg gtc tgc 3384Ile Phe Thr Leu Ala Leu Cys Phe Val Pro Thr Phe Val Val Cys 1115 11201125 tac ctc cta ctg ggc ctg gac ata cgc tca ggc atc ctc aac ctg 3429Tyr Leu Leu Leu Gly Leu Asp Ile Arg Ser Gly Ile Leu Asn Leu 1130 11351140 ctc tcc atc att atg atc ctc gtg gac acc atc ggc ctc atg gct 3474Leu Ser Ile Ile Met Ile Leu Val Asp Thr Ile Gly Leu Met Ala 1145 11501155 gtg tgg ggt atc agc tac aat gct gtg tcc ctc atc aac ctt gtc 3519Val Trp Gly Ile Ser Tyr Asn Ala Val Ser Leu Ile Asn Leu Val 1160 11651170 acg gca gtg ggc atg tct gtg gag ttc gtg tcc cac att acc cgg 3564Thr Ala Val Gly Met Ser Val Glu Phe Val Ser His Ile Thr Arg 1175 11801185 tcc ttt gct gta agc acc aag cct acc cgg ctg gag aga gcc aaa 3609Ser Phe Ala Val Ser Thr Lys Pro Thr Arg Leu Glu Arg Ala Lys 1190 11951200 gat gct act atc ttc atg ggc agt gcg gtg ttt gct gga gtg gcc 3654Asp Ala Thr Ile Phe Met Gly Ser Ala Val Phe Ala Gly Val Ala 1205 12101215 atg acc aac ttc ccg ggc atc ctc atc ctg ggc ttt gct cag gcc 3699Met Thr Asn Phe Pro Gly Ile Leu Ile Leu Gly Phe Ala Gln Ala 1220 12251230 cag ctt atc cag att ttc ttc ttc cgc ctc aac ctc ctg atc acc 3744Gln Leu Ile Gln Ile Phe Phe Phe Arg Leu Asn Leu Leu Ile Thr 1235 12401245 ttg ctg ggt ctg cta cac ggc ctg gtc ttc ctg ccc gtt gtc ctc 3789Leu Leu Gly Leu Leu His Gly Leu Val Phe Leu Pro Val Val Leu 1250 12551260 agc tat ctg ggg cca gat gtt aac caa gct ctg gta ctg gag gag 3834Ser Tyr Leu Gly Pro Asp Val Asn Gln Ala Leu Val Leu Glu Glu 1265 12701275 aaa cta gcc act gag gca gcc atg gtc tca gag cct tct tgc cca 3879Lys Leu Ala Thr Glu Ala Ala Met Val Ser Glu Pro Ser Cys Pro 1280 12851290 cag tac ccc ttc ccg gct gat gca aac acc agt gac tat gtt aac 3924Gln Tyr Pro Phe Pro Ala Asp Ala Asn Thr Ser Asp Tyr Val Asn 1295 13001305 tac ggc ttt aat cca gaa ttt atc cct gaa att aat gct gct agc 3969Tyr Gly Phe Asn Pro Glu Phe Ile Pro Glu Ile Asn Ala Ala Ser 1310 13151320 agc tct ctg ccc aaa agt gac caa aag ttc taa 4002 Ser Ser Leu ProLys Ser Asp Gln Lys Phe 1325 1330 12 1333 PRT Mus sp. 12 Met Ala Ala AlaTrp Gln Gly Trp Leu Leu Trp Ala Leu Leu Leu Asn 1 5 10 15 Ser Ala GlnGly Glu Leu Tyr Thr Pro Thr His Lys Ala Gly Phe Cys 20 25 30 Thr Phe TyrGlu Glu Cys Gly Lys Asn Pro Glu Leu Ser Gly Gly Leu 35 40 45 Thr Ser LeuSer Asn Ile Ser Cys Leu Ser Asn Thr Pro Ala Arg His 50 55 60 Val Thr GlyAsp His Leu Ala Leu Leu Gln Arg Val Cys Pro Arg Leu 65 70 75 80 Tyr AsnGly Pro Asn Asp Thr Tyr Ala Cys Cys Ser Thr Lys Gln Leu 85 90 95 Val SerLeu Asp Ser Ser Leu Ser Ile Thr Lys Ala Leu Leu Thr Arg 100 105 110 CysPro Ala Cys Ser Glu Asn Phe Val Ser Ile His Cys His Asn Thr 115 120 125Cys Ser Pro Asp Gln Ser Leu Phe Ile Asn Val Thr Arg Val Val Gln 130 135140 Arg Asp Pro Gly Gln Leu Pro Ala Val Val Ala Tyr Glu Ala Phe Tyr 145150 155 160 Gln Arg Ser Phe Ala Glu Lys Ala Tyr Glu Ser Cys Ser Arg ValArg 165 170 175 Ile Pro Ala Ala Ala Ser Leu Ala Val Gly Ser Met Cys GlyVal Tyr 180 185 190 Gly Ser Ala Leu Cys Asn Ala Gln Arg Trp Leu Asn PheGln Gly Asp 195 200 205 Thr Gly Asn Gly Leu Ala Pro Leu Asp Ile Thr PheHis Leu Leu Glu 210 215 220 Pro Gly Gln Ala Leu Ala Asp Gly Met Lys ProLeu Asp Gly Lys Ile 225 230 235 240 Thr Pro Cys Asn Glu Ser Gln Gly GluAsp Ser Ala Ala Cys Ser Cys 245 250 255 Gln Asp Cys Ala Ala Ser Cys ProVal Ile Pro Pro Pro Pro Ala Leu 260 265 270 Arg Pro Ser Phe Tyr Met GlyArg Met Pro Gly Trp Leu Ala Leu Ile 275 280 285 Ile Ile Phe Thr Ala ValPhe Val Leu Leu Ser Val Val Leu Val Tyr 290 295 300 Leu Arg Val Ala SerAsn Arg Asn Lys Asn Lys Thr Ala Gly Ser Gln 305 310 315 320 Glu Ala ProAsn Leu Pro Arg Lys Arg Arg Phe Ser Pro His Thr Val 325 330 335 Leu GlyArg Phe Phe Glu Ser Trp Gly Thr Arg Val Ala Ser Trp Pro 340 345 350 LeuThr Val Leu Ala Leu Ser Phe Ile Val Val Ile Ala Leu Ser Val 355 360 365Gly Leu Thr Phe Ile Glu Leu Thr Thr Asp Pro Val Glu Leu Trp Ser 370 375380 Ala Pro Lys Ser Gln Ala Arg Lys Glu Lys Ala Phe His Asp Glu His 385390 395 400 Phe Gly Pro Phe Phe Arg Thr Asn Gln Ile Phe Val Thr Ala LysAsn 405 410 415 Arg Ser Ser Tyr Lys Tyr Asp Ser Leu Leu Leu Gly Pro LysAsn Phe 420 425 430 Ser Gly Ile Leu Ser Leu Asp Leu Leu Gln Glu Leu LeuGlu Leu Gln 435 440 445 Glu Arg Leu Arg His Leu Gln Val Trp Ser His GluAla Gln Arg Asn 450 455 460 Ile Ser Leu Gln Asp Ile Cys Tyr Ala Pro LeuAsn Pro His Asn Thr 465 470 475 480 Ser Leu Thr Asp Cys Cys Val Asn SerLeu Leu Gln Tyr Phe Gln Asn 485 490 495 Asn His Thr Leu Leu Leu Leu ThrAla Asn Gln Thr Leu Asn Gly Gln 500 505 510 Thr Ser Leu Val Asp Trp LysAsp His Phe Leu Tyr Cys Ala Asn Ala 515 520 525 Pro Leu Thr Tyr Lys AspGly Thr Ala Leu Ala Leu Ser Cys Ile Ala 530 535 540 Asp Tyr Gly Ala ProVal Phe Pro Phe Leu Ala Val Gly Gly Tyr Gln 545 550 555 560 Gly Thr AspTyr Ser Glu Ala Glu Ala Leu Ile Ile Thr Phe Ser Ile 565 570 575 Asn AsnTyr Pro Ala Asp Asp Pro Arg Met Ala His Ala Lys Leu Trp 580 585 590 GluGlu Ala Phe Leu Lys Glu Met Gln Ser Phe Gln Arg Ser Thr Ala 595 600 605Asp Lys Phe Gln Ile Ala Phe Ser Ala Glu Arg Ser Leu Glu Asp Glu 610 615620 Ile Asn Arg Thr Thr Ile Gln Asp Leu Pro Val Phe Ala Ile Ser Tyr 625630 635 640 Leu Ile Val Phe Leu Tyr Ile Ser Leu Ala Leu Gly Ser Tyr SerArg 645 650 655 Trp Ser Arg Val Ala Val Asp Ser Lys Ala Thr Leu Gly LeuGly Gly 660 665 670 Val Ala Val Val Leu Gly Ala Val Val Ala Ala Met GlyPhe Tyr Ser 675 680 685 Tyr Leu Gly Val Pro Ser Ser Leu Val Ile Ile GlnVal Val Pro Phe 690 695 700 Leu Val Leu Ala Val Gly Ala Asp Asn Ile PheIle Phe Val Leu Glu 705 710 715 720 Tyr Gln Arg Leu Pro Arg Met Pro GlyGlu Gln Arg Glu Ala His Ile 725 730 735 Gly Arg Thr Leu Gly Ser Val AlaPro Ser Met Leu Leu Cys Ser Leu 740 745 750 Ser Glu Ala Ile Cys Phe PheLeu Gly Ala Leu Thr Ser Met Pro Ala 755 760 765 Val Arg Thr Phe Ala LeuThr Ser Gly Leu Ala Ile Ile Phe Asp Phe 770 775 780 Leu Leu Gln Met ThrAla Phe Val Ala Leu Leu Ser Leu Asp Ser Lys 785 790 795 800 Arg Gln GluAla Ser Arg Pro Asp Val Val Cys Cys Phe Ser Ser Arg 805 810 815 Asn LeuPro Pro Pro Lys Gln Lys Glu Gly Leu Leu Leu Cys Phe Phe 820 825 830 ArgLys Ile Tyr Thr Pro Phe Leu Leu His Arg Phe Ile Arg Pro Val 835 840 845Val Leu Leu Leu Phe Leu Val Leu Phe Gly Ala Asn Leu Tyr Leu Met 850 855860 Cys Asn Ile Ser Val Gly Leu Asp Gln Asp Leu Ala Leu Pro Lys Asp 865870 875 880 Ser Tyr Leu Ile Asp Tyr Phe Leu Phe Leu Asn Arg Tyr Leu GluVal 885 890 895 Gly Pro Pro Val Tyr Phe Asp Thr Thr Ser Gly Tyr Asn PheSer Thr 900 905 910 Glu Ala Gly Met Asn Ala Ile Cys Ser Ser Ala Gly CysGlu Ser Phe 915 920 925 Ser Leu Thr Gln Lys Ile Gln Tyr Ala Ser Glu PhePro Asn Gln Ser 930 935 940 Tyr Val Ala Ile Ala Ala Ser Ser Trp Val AspAsp Phe Ile Asp Trp 945 950 955 960 Leu Thr Pro Ser Ser Ser Cys Cys ArgIle Tyr Thr Arg Gly Pro His 965 970 975 Lys Asp Glu Phe Cys Pro Ser ThrAsp Thr Ser Phe Asn Cys Leu Lys 980 985 990 Asn Cys Met Asn Arg Thr LeuGly Pro Val Arg Pro Thr Thr Glu Gln 995 1000 1005 Phe His Lys Tyr LeuPro Trp Phe Leu Asn Asp Thr Pro Asn Ile 1010 1015 1020 Arg Cys Pro LysGly Gly Leu Ala Ala Tyr Arg Thr Ser Val Asn 1025 1030 1035 Leu Ser SerAsp Gly Gln Ile Ile Ala Ser Gln Phe Met Ala Tyr 1040 1045 1050 His LysPro Leu Arg Asn Ser Gln Asp Phe Thr Glu Ala Leu Arg 1055 1060 1065 AlaSer Arg Leu Leu Ala Ala Asn Ile Thr Ala Glu Leu Arg Lys 1070 1075 1080Val Pro Gly Thr Asp Pro Asn Phe Glu Val Phe Pro Tyr Thr Ile 1085 10901095 Ser Asn Val Phe Tyr Gln Gln Tyr Leu Thr Val Leu Pro Glu Gly 11001105 1110 Ile Phe Thr Leu Ala Leu Cys Phe Val Pro Thr Phe Val Val Cys1115 1120 1125 Tyr Leu Leu Leu Gly Leu Asp Ile Arg Ser Gly Ile Leu AsnLeu 1130 1135 1140 Leu Ser Ile Ile Met Ile Leu Val Asp Thr Ile Gly LeuMet Ala 1145 1150 1155 Val Trp Gly Ile Ser Tyr Asn Ala Val Ser Leu IleAsn Leu Val 1160 1165 1170 Thr Ala Val Gly Met Ser Val Glu Phe Val SerHis Ile Thr Arg 1175 1180 1185 Ser Phe Ala Val Ser Thr Lys Pro Thr ArgLeu Glu Arg Ala Lys 1190 1195 1200 Asp Ala Thr Ile Phe Met Gly Ser AlaVal Phe Ala Gly Val Ala 1205 1210 1215 Met Thr Asn Phe Pro Gly Ile LeuIle Leu Gly Phe Ala Gln Ala 1220 1225 1230 Gln Leu Ile Gln Ile Phe PhePhe Arg Leu Asn Leu Leu Ile Thr 1235 1240 1245 Leu Leu Gly Leu Leu HisGly Leu Val Phe Leu Pro Val Val Leu 1250 1255 1260 Ser Tyr Leu Gly ProAsp Val Asn Gln Ala Leu Val Leu Glu Glu 1265 1270 1275 Lys Leu Ala ThrGlu Ala Ala Met Val Ser Glu Pro Ser Cys Pro 1280 1285 1290 Gln Tyr ProPhe Pro Ala Asp Ala Asn Thr Ser Asp Tyr Val Asn 1295 1300 1305 Tyr GlyPhe Asn Pro Glu Phe Ile Pro Glu Ile Asn Ala Ala Ser 1310 1315 1320 SerSer Leu Pro Lys Ser Asp Gln Lys Phe 1325 1330 13 3999 DNA Mus sp.misc_feature (1)..(3999) n is g or a or t or c 13 atggcngcng cntggcarggntggytnytn tgggcnytny tnytnaayws ngcncarggn 60 garytntaya cnccnacncayaargcnggn ttytgyacnt tytaygarga rtgyggnaar 120 aayccngary tnwsnggnggnytnacnwsn ytnwsnaaya thwsntgyyt nwsnaayacn 180 ccngcnmgnc aygtnacnggngaycayytn gcnytnytnc armgngtntg yccnmgnytn 240 tayaayggnc cnaaygayacntaygcntgy tgywsnacna arcarytngt nwsnytngay 300 wsnwsnytnw snathacnaargcnytnytn acnmgntgyc cngcntgyws ngaraaytty 360 gtnwsnathc aytgycayaayacntgywsn ccngaycarw snytnttyat haaygtnacn 420 mgngtngtnc armgngayccnggncarytn ccngcngtng tngcntayga rgcnttytay 480 carmgnwsnt tygcngaraargcntaygar wsntgywsnm gngtnmgnat hccngcngcn 540 gcnwsnytng cngtnggnwsnatgtgyggn gtntayggnw sngcnytntg yaaygcncar 600 mgntggytna ayttycarggngayacnggn aayggnytng cnccnytnga yathacntty 660 cayytnytng arccnggncargcnytngcn gayggnatga arccnytnga yggnaarath 720 acnccntgya aygarwsncarggngargay wsngcngcnt gywsntgyca rgaytgygcn 780 gcnwsntgyc cngtnathccnccnccnccn gcnytnmgnc cnwsnttyta yatgggnmgn 840 atgccnggnt ggytngcnytnathathath ttyacngcng tnttygtnyt nytnwsngtn 900 gtnytngtnt ayytnmgngtngcnwsnaay mgnaayaara ayaaracngc nggnwsncar 960 gargcnccna ayytnccnmgnaarmgnmgn ttywsnccnc ayacngtnyt nggnmgntty 1020 ttygarwsnt ggggnacnmgngtngcnwsn tggccnytna cngtnytngc nytnwsntty 1080 athgtngtna thgcnytnwsngtnggnytn acnttyathg arytnacnac ngayccngtn 1140 garytntggw sngcnccnaarwsncargcn mgnaargara argcnttyca ygaygarcay 1200 ttyggnccnt tyttymgnacnaaycarath ttygtnacng cnaaraaymg nwsnwsntay 1260 aartaygayw snytnytnytnggnccnaar aayttywsng gnathytnws nytngayytn 1320 ytncargary tnytngarytncargarmgn ytnmgncayy tncargtntg gwsncaygar 1380 gcncarmgna ayathwsnytncargayath tgytaygcnc cnytnaaycc ncayaayacn 1440 wsnytnacng aytgytgygtnaaywsnytn ytncartayt tycaraayaa ycayacnytn 1500 ytnytnytna cngcnaaycaracnytnaay ggncaracnw snytngtnga ytggaargay 1560 cayttyytnt aytgygcnaaygcnccnytn acntayaarg ayggnacngc nytngcnytn 1620 wsntgyathg cngaytayggngcnccngtn ttyccnttyy tngcngtngg nggntaycar 1680 ggnacngayt aywsngargcngargcnytn athathacnt tywsnathaa yaaytayccn 1740 gcngaygayc cnmgnatggcncaygcnaar ytntgggarg argcnttyyt naargaratg 1800 carwsnttyc armgnwsnacngcngayaar ttycarathg cnttywsngc ngarmgnwsn 1860 ytngargayg arathaaymgnacnacnath cargayytnc cngtnttygc nathwsntay 1920 ytnathgtnt tyytntayathwsnytngcn ytnggnwsnt aywsnmgntg gwsnmgngtn 1980 gcngtngayw snaargcnacnytnggnytn ggnggngtng cngtngtnyt nggngcngtn 2040 gtngcngcna tgggnttytaywsntayytn ggngtnccnw snwsnytngt nathathcar 2100 gtngtnccnt tyytngtnytngcngtnggn gcngayaaya thttyathtt ygtnytngar 2160 taycarmgny tnccnmgnatgccnggngar carmgngarg cncayathgg nmgnacnytn 2220 ggnwsngtng cnccnwsnatgytnytntgy wsnytnwsng argcnathtg yttyttyytn 2280 ggngcnytna cnwsnatgccngcngtnmgn acnttygcny tnacnwsngg nytngcnath 2340 athttygayt tyytnytncaratgacngcn ttygtngcny tnytnwsnyt ngaywsnaar 2400 mgncargarg cnwsnmgnccngaygtngtn tgytgyttyw snwsnmgnaa yytnccnccn 2460 ccnaarcara argarggnytnytnytntgy ttyttymgna arathtayac nccnttyytn 2520 ytncaymgnt tyathmgnccngtngtnytn ytnytnttyy tngtnytntt yggngcnaay 2580 ytntayytna tgtgyaayathwsngtnggn ytngaycarg ayytngcnyt nccnaargay 2640 wsntayytna thgaytayttyytnttyytn aaymgntayy tngargtngg nccnccngtn 2700 tayttygaya cnacnwsnggntayaaytty wsnacngarg cnggnatgaa ygcnathtgy 2760 wsnwsngcng gntgygarwsnttywsnytn acncaraara thcartaygc nwsngartty 2820 ccnaaycarw sntaygtngcnathgcngcn wsnwsntggg tngaygaytt yathgaytgg 2880 ytnacnccnw snwsnwsntgytgymgnath tayacnmgng gnccncayaa rgaygartty 2940 tgyccnwsna cngayacnwsnttyaaytgy ytnaaraayt gyatgaaymg nacnytnggn 3000 ccngtnmgnc cnacnacngarcarttycay aartayytnc cntggttyyt naaygayacn 3060 ccnaayathm gntgyccnaarggnggnytn gcngcntaym gnacnwsngt naayytnwsn 3120 wsngayggnc arathathgcnwsncartty atggcntayc ayaarccnyt nmgnaaywsn 3180 cargayttya cngargcnytnmgngcnwsn mgnytnytng cngcnaayat hacngcngar 3240 ytnmgnaarg tnccnggnacngayccnaay ttygargtnt tyccntayac nathwsnaay 3300 gtnttytayc arcartayytnacngtnytn ccngarggna thttyacnyt ngcnytntgy 3360 ttygtnccna cnttygtngtntgytayytn ytnytnggny tngayathmg nwsnggnath 3420 ytnaayytny tnwsnathathatgathytn gtngayacna thggnytnat ggcngtntgg 3480 ggnathwsnt ayaaygcngtnwsnytnath aayytngtna cngcngtngg natgwsngtn 3540 garttygtnw sncayathacnmgnwsntty gcngtnwsna cnaarccnac nmgnytngar 3600 mgngcnaarg aygcnacnathttyatgggn wsngcngtnt tygcnggngt ngcnatgacn 3660 aayttyccng gnathytnathytnggntty gcncargcnc arytnathca rathttytty 3720 ttymgnytna ayytnytnathacnytnytn ggnytnytnc ayggnytngt nttyytnccn 3780 gtngtnytnw sntayytnggnccngaygtn aaycargcny tngtnytnga rgaraarytn 3840 gcnacngarg cngcnatggtnwsngarccn wsntgyccnc artayccntt yccngcngay 3900 gcnaayacnw sngaytaygtnaaytayggn ttyaayccng arttyathcc ngarathaay 3960 gcngcnwsnw snwsnytnccnaarwsngay caraartty 3999 14 20 DNA Artificial sequence primer 14tcttcaccct tgctctttgc 20 15 24 DNA Artificial Sequence primer 15aatgatggag agtaggttga ggat 24 16 26 DNA Artificial Sequence primer 16tgcccacctt tgttgtctgc taccta 26 17 21 DNA Artificial Sequence primer 17atcgctgaca ggatgcagaa g 21 18 22 DNA Artificial Sequence primer 18tcaggaggag caatgatctt ga 22 19 30 DNA Artificial Sequence primer 19agattactgc cctggctcct agcaccatta 30 20 20 DNA Artificial Sequence primer20 atcctcatcc tgggctttgc 20 21 21 DNA Artificial Sequence primer 21gcaaggtgat caggaggttg a 21 22 29 DNA Artificial Sequence primer 22cccagcttat ccagattttc ttcttccgc 29 23 20 DNA Artificial Sequence primer23 tcttcaccct tgctctttgc 20 24 24 DNA Artificial Sequence primer 24aatgatggag agtaggttga ggat 24 25 24 DNA Artificial Sequence primer 25tgcccacctt tgttgtctgc tacc 24 26 23 DNA Artificial Sequence primer 26agcacctgtc cactgaagat ttc 23 27 21 DNA Artificial Sequence primer 27tggacgctga gcttcagttc t 21 28 24 DNA Artificial Sequence primer 28cttctctgcg ctgcctcgat ggaa 24 29 21 DNA Artificial Sequence primer 29agtaaaaagg gctcgcagga t 21 30 21 DNA Artificial Sequence primer 30ggcagctggt gacatcagag a 21 31 25 DNA Artificial Sequence primer 31aggaggccat gcaggcctac tctga 25 32 21 DNA Artificial Sequence primer 32gagtccacgg tcagtccatg t 21 33 23 DNA Artificial Sequence primer 33ttatgaacaa caatgccaag caa 23 34 34 DNA Artificial Sequence primer 34agtccttagg tagtggctta gtccctggaa gctc 34 35 52 DNA Artificial Sequenceprobe 35 gtaatacgac tcactatagg gccctgacgg tccttcctga gggaatcttc ac 52 3650 DNA Artificial Sequence probe 36 gtaatacgac tcactatagg gcctgggaagttggtcatgg ccactccagc 50 37 8 PRT Artificial Sequence FLAG tag 37 AspTyr Lys Asp Asp Asp Asp Lys 1 5 38 4 PRT Artificial Sequence motif 38Tyr Gln Arg Leu 1 39 19 PRT Artificial Sequence antigen 39 Glu Gln PheHis Lys Tyr Leu Pro Trp Phe Leu Asn Asp Pro Pro Asn 1 5 10 15 Ile ArgCys 40 16 PRT Artificial Sequence antigen 40 Glu Ala Phe Tyr Gln Arg SerPhe Ala Glu Lys Ala Tyr Glu Ser Cys 1 5 10 15 41 15 PRT ArtificialSequence antigen 41 Gly Gln Thr Ser Leu Val Asp Trp Lys Asp His Phe LeuTyr Cys 1 5 10 15 42 17 PRT Artificial Sequence antigen 42 Cys Ala AsnAla Pro Leu Thr Phe Lys Asp Gly Thr Ala Leu Ala Leu 1 5 10 15 Ser 435092 DNA Homo sapiens CDS (57)..(4136) 43 cttggctgtt cctgaggcctggcctggctc cccgctgacc ccttcccaga cctggg atg 59 Met 1 gcg gag gcc ggc ctgagg ggc tgg ctg ctg tgg gcc ctg ctc ctg cgc 107 Ala Glu Ala Gly Leu ArgGly Trp Leu Leu Trp Ala Leu Leu Leu Arg 5 10 15 ttg gcc cag agt gag ccttac aca acc atc cac cag cct ggc tac tgc 155 Leu Ala Gln Ser Glu Pro TyrThr Thr Ile His Gln Pro Gly Tyr Cys 20 25 30 gcc ttc tat gac gaa tgt gggaag aac cca gag ctg tct gga agc ctc 203 Ala Phe Tyr Asp Glu Cys Gly LysAsn Pro Glu Leu Ser Gly Ser Leu 35 40 45 atg aca ctc tcc aac gtg tcc tgcctg tcc aac acg ccg gcc cgc aag 251 Met Thr Leu Ser Asn Val Ser Cys LeuSer Asn Thr Pro Ala Arg Lys 50 55 60 65 atc aca ggt gat cac ctg atc ctatta cag aag atc tgc ccc cgc ctc 299 Ile Thr Gly Asp His Leu Ile Leu LeuGln Lys Ile Cys Pro Arg Leu 70 75 80 tac acc ggc ccc aac acc caa gcc tgctgc tcc gcc aag cag ctg gta 347 Tyr Thr Gly Pro Asn Thr Gln Ala Cys CysSer Ala Lys Gln Leu Val 85 90 95 tca ctg gaa gcg agt ctg tcg atc acc aaggcc ctc ctc acc cgc tgc 395 Ser Leu Glu Ala Ser Leu Ser Ile Thr Lys AlaLeu Leu Thr Arg Cys 100 105 110 cca gcc tgc tct gac aat ttt gtg aac ctgcac tgc cac aac acg tgc 443 Pro Ala Cys Ser Asp Asn Phe Val Asn Leu HisCys His Asn Thr Cys 115 120 125 agc ccc aat cag agc ctc ttc atc aat gtgacc cgc gtg gcc cag cta 491 Ser Pro Asn Gln Ser Leu Phe Ile Asn Val ThrArg Val Ala Gln Leu 130 135 140 145 ggg gct gga caa ctc cca gct gtg gtggcc tat gag gcc ttc tac cag 539 Gly Ala Gly Gln Leu Pro Ala Val Val AlaTyr Glu Ala Phe Tyr Gln 150 155 160 cat agc ttt gcc gag cag agc tat gactcc tgc agc cgt gtg cgc gtc 587 His Ser Phe Ala Glu Gln Ser Tyr Asp SerCys Ser Arg Val Arg Val 165 170 175 cct gca gct gcc acg ctg gct gtg ggcacc atg tgt ggc gtg tat ggc 635 Pro Ala Ala Ala Thr Leu Ala Val Gly ThrMet Cys Gly Val Tyr Gly 180 185 190 tct gcc ctt tgc aat gcc cag cgc tggctc aac ttc cag gga gac aca 683 Ser Ala Leu Cys Asn Ala Gln Arg Trp LeuAsn Phe Gln Gly Asp Thr 195 200 205 ggc aat ggt ctg gcc cca ctg gac atcacc ttc cac ctc ttg gag cct 731 Gly Asn Gly Leu Ala Pro Leu Asp Ile ThrPhe His Leu Leu Glu Pro 210 215 220 225 ggc cag gcc gtg ggg agt ggg attcag cct ctg aat gag ggg gtt gca 779 Gly Gln Ala Val Gly Ser Gly Ile GlnPro Leu Asn Glu Gly Val Ala 230 235 240 cgt tgc aat gag tcc caa ggt gacgac gtg gcg acc tgc tcc tgc caa 827 Arg Cys Asn Glu Ser Gln Gly Asp AspVal Ala Thr Cys Ser Cys Gln 245 250 255 gac tgt gct gca tcc tgt cct gccata gcc cgc ccc cag gcc ctc gac 875 Asp Cys Ala Ala Ser Cys Pro Ala IleAla Arg Pro Gln Ala Leu Asp 260 265 270 tcc acc ttc tac ctg ggc cag atgccg ggc agt ctg gtc ctc atc atc 923 Ser Thr Phe Tyr Leu Gly Gln Met ProGly Ser Leu Val Leu Ile Ile 275 280 285 atc ctc tgc tct gtc ttc gct gtggtc acc atc ctg ctt gtg gga ttc 971 Ile Leu Cys Ser Val Phe Ala Val ValThr Ile Leu Leu Val Gly Phe 290 295 300 305 cgt gtg gcc ccc gcc agg gacaaa agc aag atg gtg gac ccc aag aag 1019 Arg Val Ala Pro Ala Arg Asp LysSer Lys Met Val Asp Pro Lys Lys 310 315 320 ggc acc agc ctc tct gac aagctc agc ttc tcc acc cac acc ctc ctt 1067 Gly Thr Ser Leu Ser Asp Lys LeuSer Phe Ser Thr His Thr Leu Leu 325 330 335 ggc cag ttc ttc cag ggc tggggc acg tgg gtg gct tcg tgg cct ctg 1115 Gly Gln Phe Phe Gln Gly Trp GlyThr Trp Val Ala Ser Trp Pro Leu 340 345 350 acc atc ttg gtg cta tct gtcatc ccg gtg gtg gcc ttg gca gcg ggc 1163 Thr Ile Leu Val Leu Ser Val IlePro Val Val Ala Leu Ala Ala Gly 355 360 365 ctg gtc ttt aca gaa ctc actacg gac ccc gtg gag ctg tgg tcg gcc 1211 Leu Val Phe Thr Glu Leu Thr ThrAsp Pro Val Glu Leu Trp Ser Ala 370 375 380 385 ccc aac agc caa gcc cggagt gag aaa gct ttc cat gac cag cat ttc 1259 Pro Asn Ser Gln Ala Arg SerGlu Lys Ala Phe His Asp Gln His Phe 390 395 400 ggc ccc ttc ttc cga accaac cag gtg atc ctg acg gct cct aac cgg 1307 Gly Pro Phe Phe Arg Thr AsnGln Val Ile Leu Thr Ala Pro Asn Arg 405 410 415 tcc agc tac agg tat gactct ctg ctg ctg ggg ccc aag aac ttc agc 1355 Ser Ser Tyr Arg Tyr Asp SerLeu Leu Leu Gly Pro Lys Asn Phe Ser 420 425 430 gga atc ctg gac ctg gacttg ctg ctg gag ctg cta gag ctg cag gag 1403 Gly Ile Leu Asp Leu Asp LeuLeu Leu Glu Leu Leu Glu Leu Gln Glu 435 440 445 agg ctg cgg cac ctc caggta tgg tcg ccc gaa gca cag cgc aac atc 1451 Arg Leu Arg His Leu Gln ValTrp Ser Pro Glu Ala Gln Arg Asn Ile 450 455 460 465 tcc ctg cag gac atctgc tac gcc ccc ctc aat ccg gac aat acc agt 1499 Ser Leu Gln Asp Ile CysTyr Ala Pro Leu Asn Pro Asp Asn Thr Ser 470 475 480 ctc tac gac tgc tgcatc aac agc ctc ctg cag tat ttc cag aac aac 1547 Leu Tyr Asp Cys Cys IleAsn Ser Leu Leu Gln Tyr Phe Gln Asn Asn 485 490 495 cgc acg ctc ctg ctgctc aca gcc aac cag aca ctg atg ggg cag acc 1595 Arg Thr Leu Leu Leu LeuThr Ala Asn Gln Thr Leu Met Gly Gln Thr 500 505 510 tcc caa gtc gac tggaag gac cat ttt ctg tac tgt gcc aat gcc ccg 1643 Ser Gln Val Asp Trp LysAsp His Phe Leu Tyr Cys Ala Asn Ala Pro 515 520 525 ctc acc ttc aag gatggc aca gcc ctg gcc ctg agc tgc atg gct gac 1691 Leu Thr Phe Lys Asp GlyThr Ala Leu Ala Leu Ser Cys Met Ala Asp 530 535 540 545 tac ggg gcc cctgtc ttc ccc ttc ctt gcc att ggg ggg tac aaa gga 1739 Tyr Gly Ala Pro ValPhe Pro Phe Leu Ala Ile Gly Gly Tyr Lys Gly 550 555 560 aag gac tat tctgag gca gag gcc ctg atc atg acg ttc tcc ctc aac 1787 Lys Asp Tyr Ser GluAla Glu Ala Leu Ile Met Thr Phe Ser Leu Asn 565 570 575 aat tac cct gccggg gac ccc cgt ctg gcc cag gcc aag ctg tgg gag 1835 Asn Tyr Pro Ala GlyAsp Pro Arg Leu Ala Gln Ala Lys Leu Trp Glu 580 585 590 gag gcc ttc ttagag gaa atg cga gcc ttc cag cgt cgg atg gct ggc 1883 Glu Ala Phe Leu GluGlu Met Arg Ala Phe Gln Arg Arg Met Ala Gly 595 600 605 atg ttc cag gtcacg ttc atg gct gag cgc tct ctg gaa gac gag atc 1931 Met Phe Gln Val ThrPhe Met Ala Glu Arg Ser Leu Glu Asp Glu Ile 610 615 620 625 aat cgc accaca gct gaa gac ctg ccc atc ttt gcc acc agc tac att 1979 Asn Arg Thr ThrAla Glu Asp Leu Pro Ile Phe Ala Thr Ser Tyr Ile 630 635 640 gtc ata ttcctg tac atc tct ctg gcc ctg ggc agc tat tcc agc tgg 2027 Val Ile Phe LeuTyr Ile Ser Leu Ala Leu Gly Ser Tyr Ser Ser Trp 645 650 655 agc cga gtgatg gtg gac tcc aag gcc acg ctg ggc ctc ggc ggg gtg 2075 Ser Arg Val MetVal Asp Ser Lys Ala Thr Leu Gly Leu Gly Gly Val 660 665 670 gcc gtg gtcctg gga gca gtc atg gct gcc atg ggc ttc ttc tcc tac 2123 Ala Val Val LeuGly Ala Val Met Ala Ala Met Gly Phe Phe Ser Tyr 675 680 685 ttg ggt atccgc tcc tcc ctg gtc atc ctg caa gtg gtt cct ttc ctg 2171 Leu Gly Ile ArgSer Ser Leu Val Ile Leu Gln Val Val Pro Phe Leu 690 695 700 705 gtg ctgtcc gtg ggg gct gat aac atc ttc atc ttt gtt ctc gag tac 2219 Val Leu SerVal Gly Ala Asp Asn Ile Phe Ile Phe Val Leu Glu Tyr 710 715 720 cag aggctg ccc cgg agg cct ggg gag cca cga gag gtc cac att ggg 2267 Gln Arg LeuPro Arg Arg Pro Gly Glu Pro Arg Glu Val His Ile Gly 725 730 735 cga gcccta ggc agg gtg gct ccc agc atg ctg ttg tgc agc ctc tct 2315 Arg Ala LeuGly Arg Val Ala Pro Ser Met Leu Leu Cys Ser Leu Ser 740 745 750 gag gccatc tgc ttc ttc cta ggg gcc ctg acc ccc atg cca gct gtg 2363 Glu Ala IleCys Phe Phe Leu Gly Ala Leu Thr Pro Met Pro Ala Val 755 760 765 cgg accttt gcc ctg acc tct ggc ctt gca gtg atc ctt gac ttc ctc 2411 Arg Thr PheAla Leu Thr Ser Gly Leu Ala Val Ile Leu Asp Phe Leu 770 775 780 785 ctgcag atg tca gcc ttt gtg gcc ctg ctc tcc ctg gac agc aag agg 2459 Leu GlnMet Ser Ala Phe Val Ala Leu Leu Ser Leu Asp Ser Lys Arg 790 795 800 caggag gcc tcc cgg ttg gac gtc tgc tgc tgt gtc aag ccc cag gag 2507 Gln GluAla Ser Arg Leu Asp Val Cys Cys Cys Val Lys Pro Gln Glu 805 810 815 ctgccc ccg cct ggc cag gga gag ggg ctc ctg ctt ggc ttc ttc caa 2555 Leu ProPro Pro Gly Gln Gly Glu Gly Leu Leu Leu Gly Phe Phe Gln 820 825 830 aaggct tat gcc ccc ttc ctg ctg cac tgg atc act cga ggt gtt gtg 2603 Lys AlaTyr Ala Pro Phe Leu Leu His Trp Ile Thr Arg Gly Val Val 835 840 845 ctgctg ctg ttt ctc gcc ctg ttc gga gtg agc ctc tac tcc atg tgc 2651 Leu LeuLeu Phe Leu Ala Leu Phe Gly Val Ser Leu Tyr Ser Met Cys 850 855 860 865cac atc agc gtg gga ctg gac cag gag ctg gcc ctg ccc aag gac tcg 2699 HisIle Ser Val Gly Leu Asp Gln Glu Leu Ala Leu Pro Lys Asp Ser 870 875 880tac ctg ctt gac tat ttc ctc ttt ctg aac cgc tac ttc gag gtg ggg 2747 TyrLeu Leu Asp Tyr Phe Leu Phe Leu Asn Arg Tyr Phe Glu Val Gly 885 890 895gcc ccg gtg tac ttt gtt acc acc ttg ggc tac aac ttc tcc agc gag 2795 AlaPro Val Tyr Phe Val Thr Thr Leu Gly Tyr Asn Phe Ser Ser Glu 900 905 910gct ggg atg aat gcc atc tgc tcc agt gca ggc tgc aac aac ttc tcc 2843 AlaGly Met Asn Ala Ile Cys Ser Ser Ala Gly Cys Asn Asn Phe Ser 915 920 925ttc acc cag aag atc cag tat gcc aca gag ttc cct gag cag tct tac 2891 PheThr Gln Lys Ile Gln Tyr Ala Thr Glu Phe Pro Glu Gln Ser Tyr 930 935 940945 ctg gcc atc cct gcc tcc tcc tgg gtg gat gac ttc att gac tgg ctg 2939Leu Ala Ile Pro Ala Ser Ser Trp Val Asp Asp Phe Ile Asp Trp Leu 950 955960 acc ccg tcc tcc tgc tgc cgc ctt tat ata tct ggc ccc aat aag gac 2987Thr Pro Ser Ser Cys Cys Arg Leu Tyr Ile Ser Gly Pro Asn Lys Asp 965 970975 aag ttc tgc ccc tcg acc gtc aac tct ctg aac tgc cta aag aac tgc 3035Lys Phe Cys Pro Ser Thr Val Asn Ser Leu Asn Cys Leu Lys Asn Cys 980 985990 atg agc atc acg atg ggc tct gtg agg ccc tcg gtg gag cag ttc cat 3083Met Ser Ile Thr Met Gly Ser Val Arg Pro Ser Val Glu Gln Phe His 995 10001005 aag tat ctt ccc tgg ttc ctg aac gac cgg ccc aac atc aaa tgt 3128Lys Tyr Leu Pro Trp Phe Leu Asn Asp Arg Pro Asn Ile Lys Cys 1010 10151020 ccc aaa ggc ggc ctg gca gca tac agc acc tct gtg aac ttg act 3173Pro Lys Gly Gly Leu Ala Ala Tyr Ser Thr Ser Val Asn Leu Thr 1025 10301035 tca gat ggc cag gtt tta gac aca gtt gcc att ctg tca ccc agg 3218Ser Asp Gly Gln Val Leu Asp Thr Val Ala Ile Leu Ser Pro Arg 1040 10451050 ctg gag tac agt ggc aca atc tcg gct cac tgc aac ctc tac ctc 3263Leu Glu Tyr Ser Gly Thr Ile Ser Ala His Cys Asn Leu Tyr Leu 1055 10601065 ctg gat tca gcc tcc agg ttc atg gcc tat cac aag ccc ctg aaa 3308Leu Asp Ser Ala Ser Arg Phe Met Ala Tyr His Lys Pro Leu Lys 1070 10751080 aac tca cag gat tac aca gaa gct ctg cgg gca gct cga gag ctg 3353Asn Ser Gln Asp Tyr Thr Glu Ala Leu Arg Ala Ala Arg Glu Leu 1085 10901095 gca gcc aac atc act gct gac ctg cgg aaa gtg cct gga aca gac 3398Ala Ala Asn Ile Thr Ala Asp Leu Arg Lys Val Pro Gly Thr Asp 1100 11051110 ccg gct ttt gag gtc ttc ccc tac acg atc acc aat gtg ttt tat 3443Pro Ala Phe Glu Val Phe Pro Tyr Thr Ile Thr Asn Val Phe Tyr 1115 11201125 gag cag tac ctg acc atc ctc cct gag ggg ctc ttc atg ctc agc 3488Glu Gln Tyr Leu Thr Ile Leu Pro Glu Gly Leu Phe Met Leu Ser 1130 11351140 ctc tgc ctt gtg ccc acc ttc gct gtc tcc tgc ctc ctg ctg ggc 3533Leu Cys Leu Val Pro Thr Phe Ala Val Ser Cys Leu Leu Leu Gly 1145 11501155 ctg gac ctg cgc tcc ggc ctc ctc aac ctg ctc tcc att gtc atg 3578Leu Asp Leu Arg Ser Gly Leu Leu Asn Leu Leu Ser Ile Val Met 1160 11651170 atc ctc gtg gac act gtc ggc ttc atg gcc ctg tgg ggc atc agt 3623Ile Leu Val Asp Thr Val Gly Phe Met Ala Leu Trp Gly Ile Ser 1175 11801185 tac aat gct gtg tcc ctc atc aac ctg gtc tcg gcg gtg ggc atg 3668Tyr Asn Ala Val Ser Leu Ile Asn Leu Val Ser Ala Val Gly Met 1190 11951200 tct gtg gag ttt gtg tcc cac att acc cgc tcc ttt gcc atc agc 3713Ser Val Glu Phe Val Ser His Ile Thr Arg Ser Phe Ala Ile Ser 1205 12101215 acc aag ccc acc tgg ctg gag agg gcc aaa gag gcc acc atc tct 3758Thr Lys Pro Thr Trp Leu Glu Arg Ala Lys Glu Ala Thr Ile Ser 1220 12251230 atg gga agt gcg gtg ttt gca ggt gtg gcc atg acc aac ctg cct 3803Met Gly Ser Ala Val Phe Ala Gly Val Ala Met Thr Asn Leu Pro 1235 12401245 ggc atc ctt gtc ctg ggc ctc gcc aag gcc cag ctc att cag atc 3848Gly Ile Leu Val Leu Gly Leu Ala Lys Ala Gln Leu Ile Gln Ile 1250 12551260 ttc ttc ttc cgc ctc aac ctc ctg atc act ctg ctg ggc ctg ctg 3893Phe Phe Phe Arg Leu Asn Leu Leu Ile Thr Leu Leu Gly Leu Leu 1265 12701275 cat ggc ttg gtc ttc ctg ccc gtc atc ctc agc tac gtg ggg cct 3938His Gly Leu Val Phe Leu Pro Val Ile Leu Ser Tyr Val Gly Pro 1280 12851290 gac gtt aac ccg gct ctg gca ctg gag cag aag cgg gct gag gag 3983Asp Val Asn Pro Ala Leu Ala Leu Glu Gln Lys Arg Ala Glu Glu 1295 13001305 gcg gtg gca gca gtc atg gtg gcc tct tgc cca aat cac ccc tcc 4028Ala Val Ala Ala Val Met Val Ala Ser Cys Pro Asn His Pro Ser 1310 13151320 cga gtc tcc aca gct gac aac atc tat gtc aac cac agc ttt gaa 4073Arg Val Ser Thr Ala Asp Asn Ile Tyr Val Asn His Ser Phe Glu 1325 13301335 ggt tct atc aaa ggt gct ggt gcc atc agc aac ttc ttg ccc aac 4118Gly Ser Ile Lys Gly Ala Gly Ala Ile Ser Asn Phe Leu Pro Asn 1340 13451350 aat ggg cgg cag ttc tga tacagccaga ggccctgtct aggctctatg 4166 AsnGly Arg Gln Phe 1355 gccctgaacc aaagggttat ggggatcttc cttgtgactgccccttgaca cacgccctcc 4226 tcaaatccta ggggaggcca ttcccatgag actgcctgtcactggaggat ggcctgctct 4286 tgaggtatcc aggcagcacc actgatggct cctctgctcccatagtgggt ccccagtttc 4346 caagtcacct aggccttggg cagtgcctcc tcctgggcctgggtctggaa gttggcagga 4406 acagacacac tccatgtttg tcccacactc actcactttcctaggagccc acttctcatc 4466 caacttttcc cttctcagtt cctctctcga aagtcttaattctgtgtcag taagtcttta 4526 acacgtagca gtgtccctga gaacacagac aatgaccactaccctgggtg tgatatcaca 4586 ggaggccaga gagaggcaaa ggctcaggcc aagagccaacgctgtgggag gccggtcggc 4646 agccactccc tccagggcgc acctgcaggt ctgccatccacggccttttc tggcaagaga 4706 agggcccagg aaggatgctc tcataaggcc caggaaggatgctctcataa gcaccttggt 4766 catggattag cccctcctgg aaaatggtgt tgggtttggtctccagctcc aatacttatt 4826 aaggctgttg ctgccagtca aggccaccca ggagtctgaaggctgggagc tcttggggct 4886 gggctggtcc tcccatcttc acctcgggcc tggatcccaggcctcaaacc agcccaaccc 4946 gagcttttgg acagctctcc agaagcatga actgcagtggagatgaagat cctggctctg 5006 tgctgtgcac ataggtgttt aataaacatt tgttggcagaaaaaaaaaaa aaaaaaaaaa 5066 aaaaaaaaaa aaaaaaaaaa aaaaaa 5092 44 1359 PRTHomo sapiens 44 Met Ala Glu Ala Gly Leu Arg Gly Trp Leu Leu Trp Ala LeuLeu Leu 1 5 10 15 Arg Leu Ala Gln Ser Glu Pro Tyr Thr Thr Ile His GlnPro Gly Tyr 20 25 30 Cys Ala Phe Tyr Asp Glu Cys Gly Lys Asn Pro Glu LeuSer Gly Ser 35 40 45 Leu Met Thr Leu Ser Asn Val Ser Cys Leu Ser Asn ThrPro Ala Arg 50 55 60 Lys Ile Thr Gly Asp His Leu Ile Leu Leu Gln Lys IleCys Pro Arg 65 70 75 80 Leu Tyr Thr Gly Pro Asn Thr Gln Ala Cys Cys SerAla Lys Gln Leu 85 90 95 Val Ser Leu Glu Ala Ser Leu Ser Ile Thr Lys AlaLeu Leu Thr Arg 100 105 110 Cys Pro Ala Cys Ser Asp Asn Phe Val Asn LeuHis Cys His Asn Thr 115 120 125 Cys Ser Pro Asn Gln Ser Leu Phe Ile AsnVal Thr Arg Val Ala Gln 130 135 140 Leu Gly Ala Gly Gln Leu Pro Ala ValVal Ala Tyr Glu Ala Phe Tyr 145 150 155 160 Gln His Ser Phe Ala Glu GlnSer Tyr Asp Ser Cys Ser Arg Val Arg 165 170 175 Val Pro Ala Ala Ala ThrLeu Ala Val Gly Thr Met Cys Gly Val Tyr 180 185 190 Gly Ser Ala Leu CysAsn Ala Gln Arg Trp Leu Asn Phe Gln Gly Asp 195 200 205 Thr Gly Asn GlyLeu Ala Pro Leu Asp Ile Thr Phe His Leu Leu Glu 210 215 220 Pro Gly GlnAla Val Gly Ser Gly Ile Gln Pro Leu Asn Glu Gly Val 225 230 235 240 AlaArg Cys Asn Glu Ser Gln Gly Asp Asp Val Ala Thr Cys Ser Cys 245 250 255Gln Asp Cys Ala Ala Ser Cys Pro Ala Ile Ala Arg Pro Gln Ala Leu 260 265270 Asp Ser Thr Phe Tyr Leu Gly Gln Met Pro Gly Ser Leu Val Leu Ile 275280 285 Ile Ile Leu Cys Ser Val Phe Ala Val Val Thr Ile Leu Leu Val Gly290 295 300 Phe Arg Val Ala Pro Ala Arg Asp Lys Ser Lys Met Val Asp ProLys 305 310 315 320 Lys Gly Thr Ser Leu Ser Asp Lys Leu Ser Phe Ser ThrHis Thr Leu 325 330 335 Leu Gly Gln Phe Phe Gln Gly Trp Gly Thr Trp ValAla Ser Trp Pro 340 345 350 Leu Thr Ile Leu Val Leu Ser Val Ile Pro ValVal Ala Leu Ala Ala 355 360 365 Gly Leu Val Phe Thr Glu Leu Thr Thr AspPro Val Glu Leu Trp Ser 370 375 380 Ala Pro Asn Ser Gln Ala Arg Ser GluLys Ala Phe His Asp Gln His 385 390 395 400 Phe Gly Pro Phe Phe Arg ThrAsn Gln Val Ile Leu Thr Ala Pro Asn 405 410 415 Arg Ser Ser Tyr Arg TyrAsp Ser Leu Leu Leu Gly Pro Lys Asn Phe 420 425 430 Ser Gly Ile Leu AspLeu Asp Leu Leu Leu Glu Leu Leu Glu Leu Gln 435 440 445 Glu Arg Leu ArgHis Leu Gln Val Trp Ser Pro Glu Ala Gln Arg Asn 450 455 460 Ile Ser LeuGln Asp Ile Cys Tyr Ala Pro Leu Asn Pro Asp Asn Thr 465 470 475 480 SerLeu Tyr Asp Cys Cys Ile Asn Ser Leu Leu Gln Tyr Phe Gln Asn 485 490 495Asn Arg Thr Leu Leu Leu Leu Thr Ala Asn Gln Thr Leu Met Gly Gln 500 505510 Thr Ser Gln Val Asp Trp Lys Asp His Phe Leu Tyr Cys Ala Asn Ala 515520 525 Pro Leu Thr Phe Lys Asp Gly Thr Ala Leu Ala Leu Ser Cys Met Ala530 535 540 Asp Tyr Gly Ala Pro Val Phe Pro Phe Leu Ala Ile Gly Gly TyrLys 545 550 555 560 Gly Lys Asp Tyr Ser Glu Ala Glu Ala Leu Ile Met ThrPhe Ser Leu 565 570 575 Asn Asn Tyr Pro Ala Gly Asp Pro Arg Leu Ala GlnAla Lys Leu Trp 580 585 590 Glu Glu Ala Phe Leu Glu Glu Met Arg Ala PheGln Arg Arg Met Ala 595 600 605 Gly Met Phe Gln Val Thr Phe Met Ala GluArg Ser Leu Glu Asp Glu 610 615 620 Ile Asn Arg Thr Thr Ala Glu Asp LeuPro Ile Phe Ala Thr Ser Tyr 625 630 635 640 Ile Val Ile Phe Leu Tyr IleSer Leu Ala Leu Gly Ser Tyr Ser Ser 645 650 655 Trp Ser Arg Val Met ValAsp Ser Lys Ala Thr Leu Gly Leu Gly Gly 660 665 670 Val Ala Val Val LeuGly Ala Val Met Ala Ala Met Gly Phe Phe Ser 675 680 685 Tyr Leu Gly IleArg Ser Ser Leu Val Ile Leu Gln Val Val Pro Phe 690 695 700 Leu Val LeuSer Val Gly Ala Asp Asn Ile Phe Ile Phe Val Leu Glu 705 710 715 720 TyrGln Arg Leu Pro Arg Arg Pro Gly Glu Pro Arg Glu Val His Ile 725 730 735Gly Arg Ala Leu Gly Arg Val Ala Pro Ser Met Leu Leu Cys Ser Leu 740 745750 Ser Glu Ala Ile Cys Phe Phe Leu Gly Ala Leu Thr Pro Met Pro Ala 755760 765 Val Arg Thr Phe Ala Leu Thr Ser Gly Leu Ala Val Ile Leu Asp Phe770 775 780 Leu Leu Gln Met Ser Ala Phe Val Ala Leu Leu Ser Leu Asp SerLys 785 790 795 800 Arg Gln Glu Ala Ser Arg Leu Asp Val Cys Cys Cys ValLys Pro Gln 805 810 815 Glu Leu Pro Pro Pro Gly Gln Gly Glu Gly Leu LeuLeu Gly Phe Phe 820 825 830 Gln Lys Ala Tyr Ala Pro Phe Leu Leu His TrpIle Thr Arg Gly Val 835 840 845 Val Leu Leu Leu Phe Leu Ala Leu Phe GlyVal Ser Leu Tyr Ser Met 850 855 860 Cys His Ile Ser Val Gly Leu Asp GlnGlu Leu Ala Leu Pro Lys Asp 865 870 875 880 Ser Tyr Leu Leu Asp Tyr PheLeu Phe Leu Asn Arg Tyr Phe Glu Val 885 890 895 Gly Ala Pro Val Tyr PheVal Thr Thr Leu Gly Tyr Asn Phe Ser Ser 900 905 910 Glu Ala Gly Met AsnAla Ile Cys Ser Ser Ala Gly Cys Asn Asn Phe 915 920 925 Ser Phe Thr GlnLys Ile Gln Tyr Ala Thr Glu Phe Pro Glu Gln Ser 930 935 940 Tyr Leu AlaIle Pro Ala Ser Ser Trp Val Asp Asp Phe Ile Asp Trp 945 950 955 960 LeuThr Pro Ser Ser Cys Cys Arg Leu Tyr Ile Ser Gly Pro Asn Lys 965 970 975Asp Lys Phe Cys Pro Ser Thr Val Asn Ser Leu Asn Cys Leu Lys Asn 980 985990 Cys Met Ser Ile Thr Met Gly Ser Val Arg Pro Ser Val Glu Gln Phe 9951000 1005 His Lys Tyr Leu Pro Trp Phe Leu Asn Asp Arg Pro Asn Ile Lys1010 1015 1020 Cys Pro Lys Gly Gly Leu Ala Ala Tyr Ser Thr Ser Val AsnLeu 1025 1030 1035 Thr Ser Asp Gly Gln Val Leu Asp Thr Val Ala Ile LeuSer Pro 1040 1045 1050 Arg Leu Glu Tyr Ser Gly Thr Ile Ser Ala His CysAsn Leu Tyr 1055 1060 1065 Leu Leu Asp Ser Ala Ser Arg Phe Met Ala TyrHis Lys Pro Leu 1070 1075 1080 Lys Asn Ser Gln Asp Tyr Thr Glu Ala LeuArg Ala Ala Arg Glu 1085 1090 1095 Leu Ala Ala Asn Ile Thr Ala Asp LeuArg Lys Val Pro Gly Thr 1100 1105 1110 Asp Pro Ala Phe Glu Val Phe ProTyr Thr Ile Thr Asn Val Phe 1115 1120 1125 Tyr Glu Gln Tyr Leu Thr IleLeu Pro Glu Gly Leu Phe Met Leu 1130 1135 1140 Ser Leu Cys Leu Val ProThr Phe Ala Val Ser Cys Leu Leu Leu 1145 1150 1155 Gly Leu Asp Leu ArgSer Gly Leu Leu Asn Leu Leu Ser Ile Val 1160 1165 1170 Met Ile Leu ValAsp Thr Val Gly Phe Met Ala Leu Trp Gly Ile 1175 1180 1185 Ser Tyr AsnAla Val Ser Leu Ile Asn Leu Val Ser Ala Val Gly 1190 1195 1200 Met SerVal Glu Phe Val Ser His Ile Thr Arg Ser Phe Ala Ile 1205 1210 1215 SerThr Lys Pro Thr Trp Leu Glu Arg Ala Lys Glu Ala Thr Ile 1220 1225 1230Ser Met Gly Ser Ala Val Phe Ala Gly Val Ala Met Thr Asn Leu 1235 12401245 Pro Gly Ile Leu Val Leu Gly Leu Ala Lys Ala Gln Leu Ile Gln 12501255 1260 Ile Phe Phe Phe Arg Leu Asn Leu Leu Ile Thr Leu Leu Gly Leu1265 1270 1275 Leu His Gly Leu Val Phe Leu Pro Val Ile Leu Ser Tyr ValGly 1280 1285 1290 Pro Asp Val Asn Pro Ala Leu Ala Leu Glu Gln Lys ArgAla Glu 1295 1300 1305 Glu Ala Val Ala Ala Val Met Val Ala Ser Cys ProAsn His Pro 1310 1315 1320 Ser Arg Val Ser Thr Ala Asp Asn Ile Tyr ValAsn His Ser Phe 1325 1330 1335 Glu Gly Ser Ile Lys Gly Ala Gly Ala IleSer Asn Phe Leu Pro 1340 1345 1350 Asn Asn Gly Arg Gln Phe 1355 45 4471DNA Mus musculus 45 ggatcacttc ctggctctgg gatggcagct gcctggcagggatggctgct ctgggccctg 60 ctcctgaatt cggcccaggg tgagctctac acacccactcacaaagctgg cttctgcacc 120 ttttatgaag agtgtgggaa gaacccagag ctttctggaggcctcacatc actatccaat 180 atctcctgct tgtctaatac cccagccccg ccatgtcacaggtgaccacc tggctcttct 240 ccagcgcgtc tgtccccgcc tatacaatgg ccccaatgacacctatgcct gttgctctac 300 caagcagctg gtgtcattag acagtagcct gtctatcaccaaggccctcc ttacacgctg 360 cccggcatgc tctgaaaatt ttgtgagcat acactgtcataatacctgca gccctgacca 420 gagcctcttc atcaatgtta ctcgcgtggt tcagcgggaccctggacagc ttcctgctgt 480 ggtggcctat gaggcctttt atcaacgcag ttttgcagagaaggcctatg agtcctgtag 540 ccgggtgcgc atccctgcag ctgcctcgct ggctgtgggcagcatgtgtg gagtgtatgg 600 ctctgccctc tgcaatgctc agcgcctggc tcaacttccaaggagacaca gggaatggcc 660 tggctccgct ggacatcacc ttccacctct tggagcctggccaggccctg gcagatggga 720 tgaagccact ggatgggaag atcaaaccct gcaatgagtcccagggtgaa gactcggcag 780 cctgttcctg ccaggactgt gcagcatcct gccctgtcatccctccgccc ccggccctgc 840 gcccttcttt ctacatgggt cgaatgccag gctggctggctctcatcatc atcttcactg 900 ctgtctttgt attgctctct gttgtccttg tgtatctccgagtggcttcc aacaggaaca 960 agaacaagac agcaggctcc caggaagccc ccaacctccctcgtaagcgc agattctcac 1020 ctcacactgt ccttggccgg ttcttcgaga gctggggaacaatggtggcc tcatggccac 1080 tcactgtctt ggcactgtcc ttcatagttg tgatagccttgtcagtaggc ctgaccttta 1140 tagaactcac cacagaccct gtggaactgt ggtcggcccctaaaagccaa gcccggaaag 1200 aaaaggcttt ccatgacgag cattttggcc ccttcttccgaaccaaccag atttttgtga 1260 cagctaagaa caggtccagc tacaagtacg actccctgctgctagggccc aagaacttca 1320 gtgggatcct atccctggac ttgctgcagg agctgttggagctacaggag agacttcgac 1380 acctgcaagt gtggtcccat gaggcacagc gcaacatctccctccaggac atctgctatg 1440 ctcccctcaa accgcataac accagcctca ctgactgctgtgtcaacagc ctccttcaat 1500 acttccagaa caaccacaca ctcctgctgc tcacagccaaccagactctg aatggccaga 1560 cctccctggt ggactggaag gaccatttcc tctactgtgccaatgcccct ctcacgtaca 1620 aagatggcac agccctggcc ctgagctgca tagctgactacggggcgcct gtcttcccct 1680 tccttgctgt tgggggctac caagggacgg actactcggaggcagaagcc ctgatcataa 1740 ccttctctat caataactac cccgctgatg atccccgcatggcccacgcc aagctctggg 1800 aggaggcttt cttgaaggaa atgcaatcct tccagagaagcacagctgac aagttccaga 1860 ttgcgttctc agctgagcgt tctctggagg acgagatcaatcgcactacc atccaggacc 1920 tgcctgtctt tgccatcagc taccttatcg tcttcctgtacatctccctg gccctgggca 1980 gctactccag atggagccga gttgcggtgg attccaaggctactctgggc ctaggtgggg 2040 tggctgttgt gctgggagca gtcgtggctg ccatgggcttctactcctac ctgggtgtcc 2100 cctcctctct ggtcatcatt caagtggtac ctttcctggtgctggctgtg ggagctgaca 2160 acatcttcat ctttgttctt gagtaccaga ggctgcctaggatgcccggg gagcagcgag 2220 aggctcacat tggccgcacc ctgggtagtg tggcccccagcatgctgctg tgcagcctct 2280 ctgaggccat ctgcttcttt ctaggggccc tgacctccatgccagctgtg aggacctttg 2340 ccttgacctc tggcttagca atcatctttg acttcctgctccagatgaca gcctttgtgg 2400 ccctgctctc cctggatagc aagaggcagg aggcctctcgccccgacgtc gtgtgctgct 2460 tttcaagccg aaatctgccc ccaccgaaac aaaaagaaggcctcttactt tgcttcttcc 2520 gcaagatata cactcccttc ctgctgcaca gattcatccgccctgttgtg ctgctgctct 2580 ttctggtcct gtttggagca aacctctact taatgtgcaacatcagcgtg gggctggacc 2640 aggatctggc tctgcccaag gattcctacc tgatagactacttcctcttt ctgaaccggt 2700 acttggaagt ggggcctcca gtgtactttg acaccacctcaggctacaac ttttccaccg 2760 aggcaggcat gaacgccatt tgctctagtg caggctgtgagagcttctcc ctaacccaga 2820 aaatccagta tgccagtgaa ttccctaatc agtcttatgtggctattgct gcatcctcct 2880 gggtagatga cttcatcgac tggctgaccc catcctcctcctgctgccgc atttataccc 2940 gtggccccca taaagatgag ttctgtccct caacggatacttccttcaac tgtctcaaaa 3000 actgcatgaa ccgcactctg ggtcccgtga gacccacaacagaacagttt cataagtacc 3060 tgccctggtt cctgaatgat acgcccaaca tcagatgtcttaaagggggc ctagcagcgt 3120 atagaacctc tgtgaatttg atctcagatg gccagattatagcctcccag ttcatggcct 3180 accacaagcc cttacggaac tcacaggact ttacagaagctctccgggca tcccggttgc 3240 tagcagccaa catcacagct gaactacgga aggtgcctgggacagatccc aactttgagg 3300 tcttccctta cacgatctcc aatgtgttct accagcaatacctgacggtt ctccctgagg 3360 gaatcttcac tcttgctctc tgcttcgtgc ccacctttgtggtctgctac ctcctactgg 3420 gcctggacat acgctcaggc atcctcaacc tgctctccatcattatgatc ctcgtggaca 3480 ccatcggcct catggctgtg tggggtatca gctacaatgctgtgtccctc atcaaccttg 3540 tcacggcagt gggcatgtct gtggagttcg tgtcccacattacccggtcc tttgctgtaa 3600 gcaccaagcc tacccggctg gagagagcca aagatgctactatcttcatg ggcagtgcgg 3660 tgtttgctgg agtggccatg accaacttcc cgggcatcctcatcctgggc tttgctcagg 3720 cccagcttat ccagattttc ttcttccgcc tcaacctcctgatcaccttg ctgggtctgc 3780 tacacggcct ggtcttcctg cccgttgtcc tcagctatctggggccagat gttaaccaag 3840 ctctggtact ggaggagaaa ctagccactg aggcagccatggtctcagag ccttcttgcc 3900 cacagtaccc cttcccggct gatgcaaaca ccagtgacctatgttaacta aggctttaat 3960 ccagaattta tccctgaaat taatgctgct agcagctctctgcccaaaag tgaccaaaag 4020 ttctaatgga gtaggagctt gtccaggctc catggttcttgctgataagg ggccacgagg 4080 gtcttccctc tggttgtttc caaggcctgg ggaaagttgttccagaaaaa aattgctggc 4140 attcttgtcc tgaggcagcc agcactggcc actttgttgtcataggtccc cgaggccatg 4200 atcagattac ctcctctgta aagagaatat cttgagtattgtatgggatg tatcacatgt 4260 caattaaaaa ggccatggcc tatggcttag gcaggaaatagggtgtggaa catccaggag 4320 aagaaaggat tctgggataa aggacacttg ggaacgtgtggcagtggtac ctgagcacag 4380 gtaattagcc atgtggcgaa atgtagatta atataaatgcatatctaagt tatgattcta 4440 gtctagctat atggccaagg tatttataaa t 4471 46 25DNA Artificial sequence primer 46 atgttaggtg agtctgaacc taccc 25 47 25DNA Artificial sequence primer 47 ggattgcatt tccttcaaga aagcc 25 48 25DNA Artificial sequence primer 48 tatggctctg ccctctgcaa tgctc 25 49 28DNA Artificial sequence primer 49 tcagcagcct ctgttccaca tacacttc 28 5025 DNA Artificial sequence primer 50 gttccacagg gtctgtggtg agttc 25

We claim:
 1. An isolated polypeptide comprising 42 or more contiguousamino acids from an amino acid sequence selected from SEQ ID NOs: 2 and12.
 2. An isolated polypeptide comprising an amino acid sequenceselected from SEQ ID NOs: 2 and
 12. 3. An isolated polynucleotideencoding a polypeptide of claim
 1. 4. An isolated polynucleotidecomprising a nucleotide sequence selected from SEQ ID NOs: 1 and
 11. 5.A recombinant vector comprising the polynucleotide of claim
 3. 6. A hostcell comprising the vector of claim
 5. 7. An antibody which specificallybinds to a polypeptide of claim
 1. 8. An antibody which specificallybinds to a polypeptide comprising an amino acid sequence selected fromSEQ ID NOs: 39-42.
 9. A method for making a polypeptide comprisingculturing a host cell of claim 6 under conditions in which the nucleicacid is expressed.
 10. The method of claim 9 wherein the polypeptide isisolated from the culture.
 11. A method for identifying an antagonist ofNPC1L1 comprising: (a) contacting a host cell expressing a polypeptidecomprising an amino acid sequence selected from SEQ ID NOs: 2, 4 and 12or a functional fragment thereof on a cell surface, in the presence of aknown amount of detectably labeled ezetimibe, with a sample to be testedfor the presence of the antagonist; and (b) measuring the amount ofdetectably labeled ezetimibe specifically bound to the polypeptide;wherein an NPC1L1 antagonist in the sample is identified by measuringsubstantially reduced binding of the detectably labeled ezetimibe to thepolypeptide, compared to what would be measured in the absence of suchan antagonist.
 12. A method for identifying an antagonist of NPC1L1comprising: (a) placing, in an aqueous suspension, a plurality ofsupport particles, impregnated with a fluorescer, to which a host cellexpressing a polypeptide comprising an amino acid sequence selected fromSEQ ID NOs: 2, 4 and 12 or a functional fragment thereof on a cellsurface are attached; (b) adding, to the suspension, radiolabeledezetimibe and a sample to be tested for the presence of the antagonist,wherein the radiolabel emits radiation energy capable of activating thefluorescer upon the binding of the ezetimibe to the polypeptide toproduce light energy, whereas radiolabeled ezetimibe that does not bindto the polypeptide is, generally, too far removed from the supportparticles to enable the radioactive energy to activate the fluorescer;and (c) measuring the light energy emitted by the fluorescer in thesuspension; wherein an NPC1L1 antagonist in the sample is identified bymeasuring substantially reduced light energy emission, compared to whatwould be measured in the absence of such an antagonist.
 13. The methodof claim 12 wherein the fluorescer is selected from yttrium silicate,yttrium oxide, diphenyloxazole and polyvinyltoluene.
 14. A method ofclaim 11 wherein the ezetimibe is labeled with a radiolabel selectedfrom ³H and ¹²⁵I.
 15. A method of claim 12 wherein the ezetimibe islabeled with a radiolabel selected from ³H and ¹²⁵I.
 16. A method foridentifying an antagonist of NPC1L1 comprising: (a) contacting a hostcell expressing a polypeptide comprising an amino acid sequence selectedfrom SEQ ID NOs: 2, 4 and 12 or a functional fragment thereof on a cellsurface with detectably labeled cholesterol and with a sample to betested for the presence of the antagonist; and (b) measuring the amountof detectably labeled cholesterol in the cell; wherein an NPC1L1antagonist in the sample is identified by measuring substantiallyreduced detectably labeled cholesterol within the host cell, compared towhat would be measured in the absence of such an antagonist.
 17. Themethod of claim 16 wherein the cholesterol is detectably labeled with aradiolabel selected from ³H and ¹²⁵I.
 18. A method according to claim 11wherein the host cell is selected from a chinese hamster ovary (CHO)cell, a J774 cell, a macrophage cell and a Caco2 cell.
 19. A methodaccording to claim 12 wherein the host cell is selected from a chinesehamster ovary (CHO) cell, a J774 cell, a macrophage cell and a Caco2cell.
 20. A method according to claim 16 wherein the host cell isselected from a chinese hamster ovary (CHO) cell, a J774 cell, amacrophage cell and a Caco2 cell.
 21. A mutant mouse comprising ahomozygous disruption of endogenous, chromosomal NPC1L1 wherein themouse does not produce any functional NPC1L1 protein.